Quantitation of rat pineal microvessels in different pineal regions and at different time points was performed using computer-aided morphometry based upon alkaline phosphatase histochemistry, with the aim of quantifying and describing distribution of pineal microvessels and investigating the effect of lightrdark cycle on their morphology. A morphometric method based on shape analysis of pineal sections was described to standardize delineation of putative regions regardless of section size or shape. The superiority of the alkaline phosphatase stained sections over conventional histological preparations in revealing microvessels was ascertained. Optimization of the simultaneous azo dye coupling method in frozen sections by using controlled fixation was described, its prospective application was advocated considering its superior morphometric accuracy in comparison with alternative procedures. Differential localization of alkaline phosphatase activity in pineal microvessels was demonstrated. The significance of the presence of a positive alkaline phosphatase reaction in pineal capillaries in the light of absence of pineal blood brain barrier was discussed.The alkaline phosphatase positive microvessels mainly consisted of capillaries (92.4%). Microvascular morphometric parameters in different pineal regions promoted the concept of a possible subdivision of pineal parenchyma into cortex and medulla.