The subcellular distribution patterns of aromatase, 5 alpha- and 5 beta-reductase in the hypothalamus/preoptic area of Japanese quail were studied using standard methods of centrifugation, and fractional constituents were identified by marker enzymes. Aromatase was concentrated 8-fold in the 100,000 g pellet (P3) along with a 3-fold enrichment in the microsomal marker NADPH-cytochrome c reductase (NCR) a result consistent with glandular tissues. In addition, aromatase was enriched 2-fold in the 11,000 g pellet (P2) and, owing to its large size, this fraction accounted for more total activity than P3. Although P2 contained the mitochondrial marker succinate dehydrogenase (SDH), treatment with Triton X-100 to solubilize membranes and release occluded enzymes increased measured NCR and the cytosol marker lactate dehydrogenase (LDH) 2- and 4-fold, respectively--evidence that this fraction was composed of mitochondria plus synaptosomes (pinched-off nerve terminals). To further explore the location of aromatase in the 11,000 g fraction, P2 was exposed to hypotonic buffer, a treatment known to cause lysis of synaptosomes, and then separated into three fractions P2a (11,000 g pellet), P2b (100,000 g pellet) and P2s (100,000 g supernatant). Aromatase colocalized with the microsomal marker NCR (13- and 4-fold increase, respectively) in the 100,000 g (P2b) pellet which was, however, devoid of mitochondrial enzyme activity. We infer from this that a significant portion of aromatase in brain is associated with smooth membranes present inside synaptosomes. 5 beta-Reductase in quail brain subfractions was enriched 6-fold in the 100,000 g supernatant together with a 4-fold enrichment in the cytosolic marker LDH.(ABSTRACT TRUNCATED AT 250 WORDS)