Cytochrome P450-dependent monooxygenases are important in the activation and detoxification of numerous insecticides. In this study, a Drosophila melanogaster Cyp6d4 null mutant was used to determine the role of this P450 in insecticide metabolism. This null mutant was generated by imprecise excision of a mobile P element located upstream to the P450 gene Cyp6d4. Comparative analysis between the non-functional mutant and relevant control strains shows that Cyp6d4 does not appear to be involved in the metabolism of chlorfenapyr, diazinon, imidacloprid, malathion, oxamyl, parathion, or pyrethrum extract, even though these insecticides are known to be activated or detoxified by P450-monooxygenases. No abnormalities in development were seen in the Cyp6d4 null mutant, indicating that Cyp6d4 is not critical for the metabolism of vital endogenous substrates.