The male-specific CYP2C13 gene has been isolated from two independent rat genomic libraries. This gene spans more than 50 kb and contains eight introns which are subject to the GT-AG rule. Two allelic forms of the CYP2C13 gene were identified. Determination of the exonic sequences revealed that one of them encodes cytochrome P450(+g) and the other encodes cytochrome P450(-g). Using allele-specific restriction enzyme sites, a good correlation between the genotype and the phenotype of CYP2C13 was shown. Nucleotide substitutions between the (+g) and the (-g) genes exist not only in the exons but also in the introns and the 5'-flanking region. Although five nucleotide differences were identified within 287 base pairs of the (+g) and (-g) 5'-flanking regions, the transcription initiation sites were identical. In addition to a canonical TATA box located 31 base pairs upstream of the start site of transcription, putative binding sites for the liver-enriched and liver-specific transcription factors HNF1/LF-B1/APF, HNF3, HNF4/AF-1, C/EBP, LAP, and eH-TF/TGT3 and the ubiquitous factors NF-1 and OTF-1 were identified.