The P450 monooxygenases of insects are important in the metabolism of numerous endogenous and exogenous compounds. However, identity of the P450 isoform(s) involved in these reactions is rarely known. A critical first step in the identification of important P450s is the cloning and sequencing of their genes. Toward this goal we report the genomic sequence of a new cytochrome P450, termed CYP6D3, from the house fly, Musca domestica. CYP6D3 is part of a P450 gene cluster located on chromosome 1 and is located upstream of a related gene, CYP6D1. The similar genetic structures of CYP6D3 and CYP6D1 (5 exons and 4 introns of similar length) suggest one of these genes may have been the result of a duplication event. The CYP6D3 deduced amino acid sequence indicates a protein with 518 amino acids and a molecular weight of 59.3 kDa. The CYP6D3 protein is most similar to house fly CYP6D1 (78%) and Cyp6D2 (56%) from Drosophila melanogaster. The deduced amino acid sequences of CYP6D3 and CYP6D1 are identical at the Helix I and heme binding regions.