The localization of two enkephalin-hydrolysing aminopeptidases i.e. aminopeptidase M (aminopeptidase N, EC 126.96.36.199) relatively insensitive to puromycin (Ki = 78 microM), and a puromycin-sensitive aminopeptidase (Ki = 1 microM) was studied in rat brain. The two aminopeptidases were differentially identified and/or localized using polyclonal anti-aminopeptidase M antibodies displaying anticatalytic activity and the inhibitors puromycin, bestatin and amastatin. Microvessels represent a major localization of cerebral aminopeptidase M as shown by the intense immunostaining of their walls in sections from various regions as well as in a fraction isolated from cerebral cortex homogenates by a sieving procedure. As compared to the starting homogenate, aminopeptidase M activity was enriched about twenty fold in this microvascular fraction. Aminopeptidase M was identified in this fraction by comparing the inhibitory potencies of antibodies and peptidase inhibitors towards the hydrolysis of [tyrosyl-3,5-3H, Met5]enkephalin to those found for the purified enzyme. A rather high aminopeptidase M activity was also localized in choroid plexuses. Following differential and gradient centrifugation analysis of cerebral cortex homogenates, aminopeptidase M activity was also enriched (by five to six fold) in fractions containing synaptic membranes. No significant soluble aminopeptidase M activity could be detected. These data suggest a dual localization of cerebral aminopeptidase M in microvessels and synaptic membranes consistent with its roles in preventing the access of circulating peptides to brain as well as in inactivating neuropeptides released from cerebral neurones. In comparison, puromycin-sensitive aminopeptidase activity, which is about 100 fold higher than aminopeptidase M activity in brain, was relatively low in microvessels and non-detectable in fractions enriched in synaptic membranes, being almost entirely restricted to soluble fractions.