P450 was purified from microsomal fractions of a strain of Saccharomyces cerevisiae which contained detectable P450 despite the disruption of CYP51A1. The P450 had a molecular mass of 58 kDa, similar to P450 51A1, and in a reconstituted assay with rabbit NADPH-P450 reductase and dilauryl phosphotidylcholine exhibited activity for conversion of ergosta-5,7-dienol into ergosterol. N-Terminal amino acid sequencing of the purified protein corresponded to the translated sequence of P450 61 which was recently identified during sequencing of chromosome XIII. This allowed the function of this family of P450 to be identified as sterol delta 22-desaturation in the pathway of ergosterol biosynthesis.