Genomic clones for Cyp4a12 and a novel member of the murine Cyp4a gene family were isolated. The novel gene, designated Cyp4a14, has a GC rich sequence immediately 5' of the transcription start site, and is similar to the rat CYP4A2 and CYP4A3 genes. The Cyp4a14 gene spans approximately 13 kb, and contains 12 exons; sequence similarity to the rat CYP4A2 gene sequence falls off 300 bp upstream from the start site. In view of the known sex-specific expression of the rat CYP4A2 gene, the expression and inducibility of Cyp4a14 was examined. The gene was highly inducible in the liver when mice were treated with the peroxisome proliferator, methylclofenapate; induction levels were low in control animals and no sex differences in expression were observed. By contrast, the Cyp4a12 RNA was highly expressed in liver and kidney of control male mice but was expressed at very low levels in liver and kidney of female mice. Testosterone treatment increased the level of this RNA in female liver slightly, and to a greater extent in the kidney of female mice. In agreement with studies on the cognate RNA, expression of Cyp4a12 protein was male-specific in the liver of control mice and extremely high inducibility of Cyp4a10 protein, with no sex differences, was also demonstrated. In view of the overlapping patterns of inducibility of the three Cyp4a genes, we investigated whether the three genes were co-localized in the genome. Two overlapping yeast artificial chromosome (YAC) clones were isolated, and the three Cyp4a genes were shown to be present on a single YAC of 220 kb. The Cyp4a genes are adjacent to the Cyp4b1 gene, with Cyp4a12 most distant from Cyp4b1. The clustering of these two gene subfamilies in the mouse was replicated in the human, where the CYPA411 and CYP4B1 genes were present in a single YAC clone of 440 kb. However, the human CYP4F2 gene was mapped to chromosome 19. Phylogenetic analysis of the CYP4 gene families demonstrated that CYP4A and CYP4B are more closely related than CYP4F.