Kinetic studies carried out over the past three decades, primarily with purified pig liver flavin-containing monooxygenase (FMO1), demonstrated that the mechanism of this flavoenzyme was distinctly different from other widely studied flavin-dependent monooxygenases in that reduction of O2 by nicotinamide-adenine-dinucleotide-phosphate reduced (NADPH) occurred before the addition of the xenobiotic substrate. Compounds bearing a soft nucleophilic heteroatom show substrate activity provided they could contact the enzyme-bound 4a-hydroperoxy flavin. Structure-activity studies suggest that in addition to nucleophilicity, size and charge of potential substrates are important parameters limiting access to the enzyme-bound hydroxylating intermediate form of the enzyme. The mechanism of FMO 1, 2, 3, and 4 are similar and differences in the substrate specificities of these isoforms can be attributed almost entirely to differences in the dimensions of the cleft or channel limiting access to the 4a-hydroperoxy flavin. While this model provides a satisfactory mechanism for the FMO catalyzed oxidation of very soft nucleophiles, it does not address another very important element of the catalytic cycle. The amine nitrogen atom is not an especially soft nucleophile readily hydroxylated by peroxides or peracids. How the enzymes convert an amine substrate to a form readily attacked by the hydroperoxy flavin is presently unknown. A complete resolution of this problem will only be possible after the tertiary structures of these enzymes are solved.