To analyze phenobarbital-inducible genes in the P450IIC subfamily, fragments of rabbit genomic DNA containing portions of the P450IIC1 and P450IIC4 genes were isolated and the DNA sequences of the 5′-flanking regions and exons were determined and compared to that of the P450IIC2 gene. The sequences have consensus TATA motifs about 20 bp from the mRNA initiation sites as determined by single-strand nuclease mapping and primer extension. Sequences similar to those for proposed liver-specific regulatory factors, HNF-1 and CCAAT, are found in the P450 5′-flanking regions. A glucocorticoid response element is present in the P450IIC2 gene but is not conserved in P450IIC1 or P450IIC4. Other sequences similar to binding sites for AP-1, the octamer binding proteins, and SPH-1 are present. Except for the TATA motif, none of the potential regulatory sequences was conserved at the same location in the different genes. The P450IIC1 and P450IIC2 genes exhibited high similarity detectable by dot matrix analysis throughout the 700 bp of sequenced 5′-flanking region while the P450IIC4 gene exhibited high similarity to both P450IIC1 and P450IIC2 only for about −150 nucleotides from the RNA initiation site. Southern analysis using probes from the 5′ region of the P450IIC4 gene suggests that there are at least three closely related members in the P450IIC4 subgroup in agreement with previous studies. After phenobarbital treatment, levels of P450IIC4 mRNA increased about fourfold, as measured by dot blot hybridization with a P450IIC4-specific oligonucleotide probe or by single-strand nuclease mapping of the 5′ end of the mRNA, indicating that the P450IIC4 gene characterized in this study is responsive to phenobarbital. Phenobarbital treatment of rabbits increased in vitro transcription of RNA for both the P450IIC1/2 and P450IIC4 gene subgroups but only slightly and transiently for the constitutive P450IIC3 gene. © 1990, Mary Ann Liebert, Inc. All rights reserved.