We studied the monoamine metabolizing mitochondrial enzyme, monoamine oxidase (MAO), in cerebral microvessels obtained from postnatally developing rats by measuring the specific binding of [3H]pargyline, an irreversible inhibitor of MAO, and the rate of oxidation of three known MAO substrates: benzylamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and tryptamine. MAO activity increased postnatally, with the greatest increase occurring in the second week and reaching a peak at 3 weeks of age. A concomitant increase in MAO of the cerebral cortex also occurred, but was several-fold less than that of cerebral microvessels. Using clorgyline and deprenyl, relatively specific inhibitors of MAO-A and MAO-B, we showed that cerebral microvessels contain both forms of MAO at all ages, but there was a major preponderance in the postnatal development of MAO-B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat microvessels after [3H]pargyline binding also showed two distinct bands of radioactivity at all ages. These two bands corresponded to molecular weights of approximately 65,000 for MAO-A and approximately 60,000 for MAO-B. SDS-PAGE results of brain microvessels obtained from 1-, 14-, and 42-day-old rats confirm the differential postnatal development of MAO-B in rat brain microvessels.