We have examined the distribution of progesterone 5 alpha-reductase activity in minces of discrete brain areas from two strains of adult ovariectomized mice known to be differentially responsive to the behavioral effects of exogenously administered 5 alpha-pregnane-3,20-dione (5 alpha-DHP). Minces of freshly dissected midbrain tegmentum (TEG), hypothalamus-preoptic area (HPOA), hippocampus (HIP), and parietal cortex (CTX) were incubated with [4-14C]progesterone in a Krebs-Henseleit bicarbonate buffer solution containing 10 mM glucose for 2 h at 37 degrees C. The [14C]5 alpha-DHP produced was identified by comparison with known standards in paper and thin-layer chromatography systems, derivative formation, and recrystallization to a constant 14C/3H ratio. We found a 6-fold difference in the extent to which various brain areas formed 5 alpha-DHP. The amount of conversion was similar for both strains of mice. When expressed as picomoles of 5 alpha-DHP formed per 2 h per 20 mg of tissue, the following order of activity was observed: TEG greater than HPOA greater than HIP greater than CTX.