3 alpha-Hydroxysteroid dehydrogenase in the brain is responsible for production of neuroactive tetrahydrosteroids that interact with the major inhibitory gamma-aminobutyric acid receptor complexes. Distribution of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain in rats was evaluated by activity assay and by Western immunoblotting using a monoclonal antibody against liver 3 alpha-hydroxysteroid dehydrogenase as the probe. The olfactory bulb was found to contain the highest level of 3 alpha-hydroxysteroid dehydrogenase activity, while moderate levels of the enzyme activity were found in other regions such as cerebellum, cerebral cortex, hypothalamus and pituitary. Some activities were found in the rest of the brain such as amygdala, brain stem, caudate putamen, cingulate cortex, hippocampus, midbrain, and thalamus. The protein levels of 3 alpha-hydroxysteroid dehydrogenase in different regions of the brain as detected by Western immunoblotting are comparable to those of the enzyme activity. No sexual dimorphism was found in either the concentration levels or the activities of the brain 3 alpha-hydroxysteroid dehydrogenase. At the time of birth, the rat brain already expresses a significant level of 3 alpha-hydroxysteroid dehydrogenase; the levels of brain 3 alpha-hydroxysteroid dehydrogenase activity in rats continue to rise during the first week after their birth, and reach a plateau thereafter.