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Heterologous expression in Escherichia coli, purification, and reconstitution of house fly P450 6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids. In addition to the epoxidation of cyclodiene insecticides demonstrated previously [Andersen et al. (1994) Biochemistry 33, 2171-2177], this cytochrome P450 was shown to epoxidize a variety of terpenoids such as farnesyl, geranyl, and neryl methyl esters, juvenile hormones I and III, and farnesal but not farnesol or farnesoic acid. P450 6A1 reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine did not metabolize alpha-pinene, limonene, of the insect growth regulators hydroprene and methoprene. The four geometric isomers of methyl farnesoate were metabolized predominantly to the 10,11-epoxides, but also the 6,7-epoxides and to the diepoxides. The 10,11-epoxide of methyl (2E,6E)-farnesoate was produced in a 3:1 ratio of the (10S) and (10R) enantiomers. Monoepoxides of methyl farnesoate were metabolized efficiently to the diepoxides. Methyl farnesoate epoxidation was strongly inhibited by a bulky substituted imidazole. The active site topology of P450 6A1 was studied by the reaction of the enzyme with phenyldiazene to form a phenyl-iron complex. Ferricyanide-induced in situ migration of the phenyl group showed formation of the N-phenylprotopor-phyrinporphyrin IX adducts in a 17:25:33:24 ratio of the NB:NA:NC:ND isomers. These experiments suggest that metabolism of xenobiotics by this P450, constitutively overexpressed in insecticide-resistant strains of the house fly, is not severely limited by stereochemically constrained access to the active site.