Stopped flow kinetic studies have been used to demonstrate three features of the enzymatic mechanism of the microsomal FAD-containing monooxygenase from hog liver. First, in contrast to the bacterial flavin-containing monooxygenases, reduction of the FAD is independent of substrate. Second, the rate of the reaction of reduced enzyme with oxygen to form the C(4a)-peroxyflavin intermediate is independent of substrate. Third, the rate of transformation of the C(4a)-peroxyflavin to oxidized FAD is substrate-dependent. These results are in agreement with the mechanism, determined by steady state kinetic studies (Poulsen, L.L., and Ziegler, D.M. (1979) J. Biol. Chem. 254, 6449-6455), which predicts that the reduced flavin reacts with oxygen before combination with substrate.