Monoamine oxidase B (MAO B) is an integral protein of the outer mitochondrial membrane that is involved in the deamination of vasoactive and neuroactive amines. The oxidation of these amine substrates requires the cofactor FAD, which is covalently bound to Cys-397 of human MAO B. Previously, Glu-34 and Tyr-44 of MAO B have been identified as residues which engage in noncovalent interactions with FAD that are required for subsequent covalent FAD binding and generation of catalytic activity. In this study, we have identified two additional residues, Arg-42 and Thr-45, which form noncovalent contacts with FAD that are prerequisite steps to the covalent attachment of FAD. Arg-42 and Thr-45, along with Tyr-44, comprise part of a highly conserved flavin binding sequence, RXY(T,S), that is found in other flavoproteins, several of which have well-defined X-ray crystal structures. We tested the roles of Arg-42 and Thr-45 in MAO B by constructing mutant MAO B cDNAs which encode amino acid substitutions at these residues and expressed the variant proteins in COS-7 cells. Substitution of Arg-42 or Thr-45 with alanine resulted in complete loss of MAO B activity and FAD incorporation. However, conservative substitutions of Arg-42 with lysine or Thr-45 with serine resulted in MAO B variants that retain both partial activity and partial FAD incorporation. These results indicate that Arg-42 and Thr-45 form critical noncovalent interactions with FAD that are required for the subsequent activation of MAO B by covalent coupling of FAD.