In this study, out of 127 samples were collected from soil, waste water, cockroach (Periplaneta americana) and urine, 55 isolates were obtained and identified as suspected Serratia spp according to the cultural and microscopical characteristics. After subjection to the biochemical tests, 48 isolates were identified as Serratia marcescens. Results of identification were confirmed using Api 20 E system. The ability of these isolates for glutaminase production was screened. Results showed that all the isolates were glutaminase producers; among them, S.marcescens isolate N1 obtained from cockroach was the most efficient in glutaminase production. Enzyme specific activity of the crude filtrate of this isolate was 1.25U/mg protein. Optimum conditions for glutaminase production by the locally isolated S.marcescens N1 in glutamine broth medium were studied. Results showed that maximum glutaminase production was achieved after supplementation of the production medium (pH8) with 0.5% of each of starch and meat extract and inoculation with a count of 2×104 CFU/ml of fresh bacterial culture and then incubation in a shaker incubator (150 rpm) at 30oC for 18 hours. Under these conditions, the specific activity of glutaminase produced in culture medium was sharply increased to 5.8U/mg. Glutaminase was produced at optimum conditions and purified in three purification steps; the first by precipitation with 25% saturation of ammonium sulfate, and the second by ion exchange chromatography using DEAE-Cellulose, and the third by gel filtration chromatography throughout Sephacryl S-200. Specific activity of the purified enzyme was 130 U/mg with 22.4 folds of purification and 56.5% recovery. Some biochemical characteristics of the purified enzyme were studied. Results showed that glutaminase consisted of one polypeptide chain with an average molecular weight of 43138.5 Dalton, optimum pH of enzyme activity and stability pH 8, optimum temperature for activity 35oC, and the enzyme was stable with full activity at a temperature ranged between (10- 30)oC. Activation energy for the purified glutaminase was 7045 calorie/mole. After investigating some kinetic properties of the purified glutaminase, results showed that the Km of glutaminase was 0.14×10-3 M while the maximum velocity was 0.015 M/min. An effect of chelating and reducing agents and heavy-metal ions on purified glutaminase activity was investigated. Results showed that a slight effect the glutaminase activity occurred after incubation with 5, 10 and 10 mM of EDTA, PMSF and sodium azide, respectively. Hence, glutaminase restored its full activity after incubation with 5mM of sodium azide, while enzyme lost most of its activity after incubation with 5 mM of cystein and 10 mM 2-mercaptoethanol. From such results, we may conclude that glutaminase is not considered to be a metalloenzyme. On the other hand, incubation of glutaminase in the presence of calcium and magnesium ions increased the remaining enzyme activity, but the full activity after incubation with 5 and 10 mM of sodium ions was reserved, while the enzyme lost most of its activity when using mercury, zink, copper and ferrous ions, respectively. Storage ability of glutaminase purified from locally isolated S.marcescens N1 in the presence of ammonium sulfate, glycine and sugars was studied. Results showed that incubation of glutaminase with 20% and 30% ammonium sulfate and 1% glycine kept the enzyme active for 20, 20 and 10 days, respectively. Glutaminase activity, on the other hand was decreased gradually after 30 days of incubation with 20% and 30% ammonium sulfate, glycine, tween- 20, glycerol, SDS, BSA, sucrose, mannitol and lactose, respectively. Results showed that one out of the 100 mutants obtained as a result of physical mutagenesis was efficient in glutaminase production. Enzyme specific activity in crude filtrate of this mutant reached 7.1 U/mg in comparison to the wild type (5.8 U/mg). On the other hand, chemical mutagenesis by (MNNG) caused no increase what so ever in enzyme specific activity. No competent mutant was obtained from any of the 100 mutants tested. Antitumor activity of the purified glutaminase was studied against cancer cell line AMN3 at different concentrations (31.5, 125, 250, 500, and 1000μg/ml). As a result, glutaminase has an inhibitory effect on growth of AMN3, and the cytotoxicity was increased by increasing the enzyme concentrations during 24 hours of exposure time, but it decreased after 48 and 72 hours of exposure time. Adversely a slight cytotoxic effect against normal cell line (REF) was detected under the same condition of exposure time and enzyme concentration.