Specific brain regions, namely, thalamus, tectum, tegmentum, cerebellum, medulla and pineal, from five proestrous rats were incubated for 30 min with [3H]progesterone. After reverse isotopic dilution analysis, the following metabolites were identified in all incubations by purification to constant specific activity, derivative formation and/or gas liquid chromatography trapping: [3H]5alpha-pregnane-3, 20-dione (10-20% of the starting substrate except pineal -- 0.7%), [3H]3alpha-hydroxy-5alpha-pregnan-20-one (1.6-3.8% except for pineal -- 0.5%) and [3H]20alpha-hydroxy-4-pregnen-3-one (0.05-0.11%). Preliminary results from the corpus collosum incubation indicated the presence of the same metabolites. Although some apparent constant specific activities were obtained for 20alpha-hydroxy-5alpha-pregnan-3-one and 5beta-pregnane-3, 20-dione, the low levels of 3H associated with these steroids did not permit a definitive identification. The results indicate the presence of at least delta1-steroid 5alpha-reductase, 3alpha-hydroxysteroid dehydrogenase and 20alpha-hydroxysteroid dehydrogenase activities with progesterone as substrate in the brain regions examined.