The codon usage bias of 110 mammalian cytochrome P-450 genes has been determined and analyzed in relation to a variety of genetic, biochemical, and physiological parameters. In those P-450 genes exhibiting biased usage the preferred codons generally do not differ among the four species examined (rat, rabbit, man, and mouse) or from the predominantly used codons identified for all sequenced genes in a recent data base analysis (Wada et al. (1992) Nucleic Acids Res. 20 (Suppl.), 2111-2118). Codon usage bias does not correlate with evolutionary relationships, evolutionary age, or with the extent of evolutionary conservation of orthologous proteins; there is no obvious correlation with the level of expression of a given P-450, with its inducibility, nor with its physiologic role; and neither the preferred codons nor the degree of bias differ for P-450s expressed in different tissues. Codon usage bias does correlate with the C+G content at the codon third position, and thus preferred codons usually end in C or G; for those P-450s for which gene sequences are available this bias also correlates with the C + G content of the intronic and flanking regions of these genes. Moreover, a lesser increase in the C + G content at the codon first and second positions is also evident in genes located in regions of high C + G content; this leads to predictable differences in the amino acid compositions of P-450 enzymes that correlate with genomic nucleotide composition and the degree of bias in codon usage.