Fragments of rabbit DNA have been cloned which encompass the gene for rabbit cytochrome P450IIC3. Cytochrome P450IIC3 is a constitutive member of the cytochrome P450IIC subfamily which contains both constitutive and phenobarbital-responsive genes. The cytochrome P450IIC3 gene spans at least 25 kbp and contains 8 introns which have 5′ GT's and 3′ AG's and are located in the same positions as in other family II genes. The 5′ flanking region contains a consensus TATA site about 25 bp from the RNA initiation site which was mapped by the primer extension method. Other potential regulatory sequences include a CCA AT sequence and sequences similar to binding sites for the liver-specific factor HNF-1 and the general transcription factors AP-1 and OCT. The gene sequence differs from the cytochrome P450IIC3 cloned cDNA sequence in only 2 of 1400 nucleotides, 1 of which results in an amino acid change. Since sequences coding for exon 1 and part of exon 2 were not present in the cloned cytochrome P450IIC3 cDNA, a 475-nucleotide fragment of the 5′ end of the cDNA was amplified by the polymerase chain reaction, cloned, and sequenced. The sequence of this cDNA was identical with the gene exon sequence. The protein sequence derived from the gene differs in nine positions from that determined directly for the protein. A comparison of the amino acid sequence by exons with other cytochrome P450IIC proteins revealed that, in general, the similarity was greater in C-terminal exons than in N-terminal ones. This trend is exaggerated in the comparison with P450IIC4/5, for which exon 8 had a high similarity of 87% compared to 65% overall and 58% for exon 6. However, divergence of nucleotides that do not result in amino acid changes was similar in exon 5 and exon 8, indicating that gene conversion was not responsible for the difference in similarity between these two exons and suggesting that conservation of protein sequence in this region may be important. © 1990, American Chemical Society. All rights reserved.