A NADH-or NADPH-dependent alkene monooxygenase (AMO) activity has been detected in cell-free extracts of the ethene-utilizing Mycobacterium E3 and Mycobacterium uurum L1. The activity was not linear with protein concentration in the assay suggesting AM0 is a multicomponent enzyme. The inhibition pattern of AM0 activity was very similar to the inhibition patterns published for the three-component soluble methane monooxygenases. Fractionation of crude extracts revealed that combination of two fractions was required to restore alkene monooxygenase activity. The first fraction was inhibited by acetylene, indicating it contained an oxygenase component. The second fraction contained reductase activity which was absent from non-induced cells. This reductase activity is probably the NADH-acceptor reductase of AMO.