The P450 CYP4A1 and CYP4A2 genes were isolated from a rat genomic library constructed in the vector lambda EMBL3 and their complete sequences were determined. The CYP4A1 and CYP4A2 genes spanned 14,144 and 10,576 bp and contained 13 and 12 exons, respectively. The CYP4A1 gene contained an additional intron that splits the exon corresponding to exon 12 of the CYP4A2 gene, resulting in a noncoding 13th exon in CYP4A1. The exon numbers of these genes were distinct among known P450 genes, and yet several intron-exon junctions along the P450 amino acid coding region were conserved with P450 genes in the CYP2, CYP11, and CYP21 gene families. On the basis of these data, the number of exons in the putative ancestral P450 gene was estimated. The evolutionary implications of this finding are discussed. No consensus TATA sequence was found upstream of either gene's transcription start site. Comparison of the CYP4A1 and CYP4A2 promoters with other genes that lack TATA boxes did not reveal any strong consensus sequence in their immediate upstream regions. However, a conserved 19-bp sequence was located at the positions of 42 and 48 bp upstream from the CYP4A1 and CYP4A2 genes' start sites, respectively. The CYP4A2 gene also contained two 378-bp direct repeats upstream from the start site; these repeats are derived from portions of the long interspersed middle repetitive element present in high copy numbers in the rat genome.