Numerous studies have indicated that progesterone metabolites, particularly 3alpha,5alpha-tetrahydroprogesterone, can potently influence multiple brain functions, e.g. they have the capacity to mediate gonadotropin regulation and various anticonvulsive, anesthetic and anxiolytic effects. These circulating progesterone metabolites are likely to represent only a fraction of the bioavailable pool of these steroids in that the central nervous system (CNS) also possesses enzymes that can synthesize these metabolites in situ. Therefore, because the ability of the CNS to produce these neuroactive progestins is an important consideration when assessing overall progestin function and metabolism, we measured the major progesterone metabolizing enzyme activities, namely the cytosolic NADPH and particulate NADH 5alpha-dihydroprogesterone 3alpha-hydroxysteroid oxidoreductase (3alpha-HSOR) and progesterone 5alpha-reductase activities in nine brain regions from random cycling and ovariectomized rats. These assays entailed the use of reverse isotopic dilution analysis and revealed that all three enzymic activities were present in each of the brain regions examined, but that these regions displayed differential patterns with regard to their levels of cytosolic and particulate 3alpha-HSOR activity. The cytosolic 3alpha-HSOR activity was highest in the olfactory bulb/tubercle and colliculi regions which were greater than levels in the hypothalamus/preoptic area and cerebellum which were greater than levels in the amygdala/striatum and hippocampus/dentate gyrus. Midbrain/thalamus, cerebral cortex and pons/medulla were different only from the olfactory bulb/tubercle and colliculi regions. The particulate 3alpha-HSOR activity was highest in the olfactory bulb/tubercle region followed by colliculi, hippocampus/dentate gyrus and pons/medulla which were greater than levels in the hypothalamus/preoptic area, cerebellum and amygdala/striatum. Cerebral cortex and midbrain/thalamus were different only from the olfactory bulb/tubercle area. The highest levels of 5alpha-reductase activity were found in the pons/medulla region followed by the colliculi, midbrain/thalamus, cerebellum and olfactory bulb/tubercle which were greater than levels in the amygdala/striatum, hippocampus/dentate gyrus, hypothalamus/preoptic area and cerebral cortex. It is interesting to note that although 5alpha-reductase may control, at least in part, substrate levels for the 3alpha-HSORs, the distribution of 5alpha-reductase activity in these nine brain regions did not correlate with 3alpha-HSOR levels. The differences in the levels of activity of these three enzymes in various brain regions suggests a role in maintaining a differential balance of the neuroactive steroid, 3alpha,5alpha-tetrahydroprogesterone, and its precursor, 5alpha-dihydroprogesterone, in various regions of the CNS.