The activity of flavin-containing monooxygenase (FMO) in rat brain microsomes was measured by fluorometrical determination of benzydamine (BZY) N-oxygenation with HPLC. The apparent Km value for the formation of BZY N-oxide from BZY by brain microsomes was similar to that by hepatic microsomal fraction or purified FMO, but the Vmax value for brain microsomes was about one-hundredth of that for hepatic microsomes. BZY N-oxygenation activity by brain microsomes was at a maximum near pH 8.5, slightly more acidic than the optimum pH for liver FMO. BZY N-oxygenation activity was inhibited completely by heat inactivation and markedly in the presence of 1 mM thiourea, but slightly in the presence of 1 mM SKF-525A, and it was only barely activated in the presence of 5 mM n-octylamine, a positive effector of liver FMO. The addition of rabbit antisera raised against rat liver FMO resulted in 30% inhibition of BZY N-oxygenation by solubilized brain microsomes. Compared with microsomes from five different brain regions, the activity was highest in microsomes of olfactory bulbs. These results show that the activity of FMO in rat brain is distinctly determined by BZY N-oxygenation.