Cytochrome P450 monooxygenases are involved in metabolism of hostplant allelochemicals by larval Lepidoptera. Biochemical purification of the P450 polypeptide induced in Papilio polyxenes (black swallowtail) larvae in response to xanthotoxin, a linear furanocoumarin, has allowed us to clone cDNAs encoding two allelic variants of the CYP6B1 locus. Expression of these alleles in lepidopteran cell lines using baculovirus expression vectors has demonstrated that both P450 isoforms metabolize substantial amounts of linear furanocoumarins, such as xanthotoxin and bergapten, but not angular furanocoumarins, such as angelicin and sphondin. These linear furanocoumarins are ubiquitous constituents of the hostplants of P. polyxenes. The efficiency of linear furanocoumarin metabolism is strongly affected by the nature of the substituents on the benzene ring; methoxy-derivatives are metabolized more efficiently than are derivatives with smaller (hydroxy-) or larger (8-O isopentenyl) groups. Metabolism of either bergapten or xanthotoxin is inhibited in the presence of the other. In addition, metabolism of linear furanocoumarins is inhibited by the presence of nonmetabolizable angular furanocoumarins, indicating that the active site of CYP6B1 binds angular furanocoumarins. The reactivities described here indicate that P. polyxenes larvae express at least two selective furanocoumarin-metabolic P450s: CYP6B1, which metabolizes a discrete set of linear furanocoumarins, and another P450, as yet unidentified, which metabolizes angular furanocoumarins more efficiently than does CYP6B1.