The soluble form of methane monooxygenase (MMO) catalyzes the reaction NAD(P)H + O2 + CH4 + H+-->NAD(P)+ + H2O + CH3OH. Many other hydrocarbons serve as adventitious substrates. MMO consists of three protein components: component B, reductase, and hydroxylase (MMOH), the active site of which contains a hydroxo-bridged dinuclear iron cluster that is the site of catalysis. Such a cluster has not been previously associated with oxygenases, and spectroscopic studies have been conducted to ascertain its structural features and accessibility. The mechanism of MMO has been investigated through the use of diagnostic chemical reactions and transient kinetics. Both approaches are consistent with a mechanism in which the diiron cluster is first reduced to the diferrous state and then reacts with O2. The O-O bond is apparently cleaved heterolytically to yield water and an [Fe(IV).Fe(IV)]=O species, which purportedly abstracts a hydrogen atom from methane to yield a substrate radical and a diiron cluster-bound hydroxyl radical. Recombination of the radicals yields the product methanol. An intermediate with the properties of the novel [Fe(IV).Fe(IV)] = O species has been trapped and characterized. This is the first such species to be isolated in biology. Meanwhile, the reductase and component B have roles in catalysis beyond simple electron transfer from NAD(P)H. These roles appear to be related to regulation of catalysis, and are mediated by the formation of specific component complexes that alter the physical and catalytic properties of MMOH at different stages of the turnover cycle.