Flavin-containing monooxygenase (FMO) was partially purified from rat brain microsomes through two successive chromatographies on columns of DEAE Sepharose and 2',5'-ADP Sepharose. The specific activity, benzydamine N-oxidation of partially purified brain FMO, was 122-fold higher than that of microsomes. A single band of 60 kDa was recognized by Western blotting analysis with anti-rat liver FMO. The Km value of brain FMO for thiourea was 4-fold lower, but that for cysteamine was 10-fold higher than that of liver FMO. The enzymatic activity for n-octylamine was detected in neither brain nor liver FMO. Kinetic analysis for neurotoxins also revealed that Km values of brain FMO for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and N-methyl TIQ (NMTIQ) were lower than those of liver FMO. These results indicate that rat brain FMO catalyzes several substrates of liver FMO involving neurotoxins, but it seems likely that the kinetic properties of brain FMO are somewhat different from those of liver FMO.