Two new cytochrome P450 cDNAs, named CYP28B1 and CYP4G13v2, and their 5'-flanking regions were cloned and sequenced from a housefly strain, ALHF. The cDNA sequences of CYP28B1 and CYP4G13v2 have open reading frames of 1449 and 1653 nucleotides encoding proteins of 483 and 551 amino acid residues, respectively. Sequence analysis shows that both CYP28B1 and CYP4G13v2 putative P450 proteins contain: (1) a highly hydrophobic N terminus; (2) a P450 protein signature motif, FXXGXRXCXG, known as the important ligand for heme binding; (3) a motif, YXXAXXXEXXR, which is a conserved P450 sequence coinciding with Helix K; and (4) a typical aromatic sequence, A(1)XXPXXA(2)XPXBA(3), which is conserved within most P450s. The 5'-flanking regions of CYP28B1 (>2kb) and CYP4G13v2 (>1 kb) were isolated from adaptor-ligated ALHF genomic DNA libraries. The transcription start points of CYP28B1 and CYP4G13v2 were mapped to 176 and 163 nucleotides upstream of the ATG translation start codon within the conserved arthropod promoter elements of TCATT and ACAGT, respectively. Possible regulatory binding sites for general transcription factors, Sp1 and AP1, were mapped in the 5' promoter regions of CYP28B1 whereas TFIID and Oct-1 were mapped in CYP4G13v2. Five conserved cis-acting elements for tissue- or cell-specific transcription regulatory factors were identified in the promoter regions of both P450 genes. A structure of five 153-nucleotide (nt) highly identical repeats and two partial repeat sequences were found in the promoter region of CYP28B1. The homologous (90% identity) sequences of the 153-nt repeat were also found in the promoter region of CYP4G13v2. The homologous sequences of the repeat in other insect P450 gene promoter regions are discussed.