The parsnip webworm, Depressaria pastinacella, a specialist on two genera in Apiaceae, feeds exclusively on the furanocoumarin-containing reproductive structures of its host plants. This caterpillar relies principally on cytochrome P450-mediated detoxification for coping with the high concentrations of furanocoumarins in its diet. A cDNA encoding the furanocoumarin-inducible P450 CYP6AB3 from this species was coexpressed with house-fly NADPH P450 reductase in baculovirus-infected Sf9 cells and tested for binding and metabolism of the six furanocoumarins typically encountered in host plant tissues. Only imperatorin and bergapten bind in close proximity to the catalytic haem and only imperatorin is metabolized (V(max) and K(m) of 2.412 pmol/min per pmol P450 and 94.28 microm, respectively). Purification of the imperatorin metabolite by normal phase HPLC and characterization of its structure by MS-MS analysis indicate that CYP6AB3 initially epoxidizes the carbon-carbon pi-bond on the isoprenyl side chain on imperatorin. An improved molecular model for the CYP6AB3 protein based on this biochemical characterization and the recently defined mammalian CYP3A4 crystal structure provides insight into the remarkable substrate specificity of this protein.