Benzo(a)pyrene hydroxylation has been observed in Saccharomyces cerevisiae and the role of sterol 14 alpha-demethylase (CYP51A1) in this activity has been examined by using a strain which contains a gene disruption of CYP51A1. This strain still contained P450 protein(s) with a Soret absorption maximum at 448nm in reduced carbon monoxide difference spectra of the microsomal fraction. On addition of benzo(a)pyrene to this microsomal extract a typical Type I substrate-binding spectrum was obtained and was also observed for the isogenic sister strain containing no CYP51A1 gene disruption. Microsomal extracts of both strains had equivalent activity in the aryl hydrocarbon hydroxylase assay. These results indicate a yeast benzo(a)pyrene hydroxylase activity distinct from sterol 14 alpha-demethylase P450.