A total of 35 sample of infants stool collected from Al-Mansor hospital for children and 20 sample of dairy products collected from Baghdad markets. Eleven isolate of Lactobacillus acidophilus were obtained from stool sample and seven isolates of also Lactobacillus acidophilus were obtained from yoghurt , the bacteria isolated and identified according to screening method and using specialized growth media. Isolates tested for their ability to produce ?-galactosidase by using O-Nitrophynel-?-D-Galactosidase (ONPG) which is lactose analog , and the ability of these isolates to enzyme production were detected in the medium by using X-gal as chromogenic substrate for ?-galactosidase which turn the color of colony from white to blue. Optimal pH and temperature for enzyme activity were determined and found that the stool isolates gave highest ?-galactosidase activity at pH 6.5 and 37?C while the yoghurt isolates gave a highest enzyme activity at pH 6.5 and 40?C . Enzyme activity was determined for all stool and yoghourt isolates under their optimal condition, and found that the yoghurt isolates is more active in enzyme production than stool isolates, and from the yoghurt isolates the most active one was selected for further study. The ability of the most active yoghurt bacterial culture for lactose consumption was estimated by using five inoculums size (1 , 1.25 , 1.5 , 1.75 , 2%) and for four incubation periods (18 , 24 , 36 , 48hr.) with defined sugar concentration (1g/50ml) , total viable count was performed after each incubation periods , and found that by inoculation with 2% and incubation for 48 hour , 28.5x10 cell/ml was able to consume about 1g of lactose.