A mutant (erg6) of Saccharomyces cerevisiae defective in S-adenosylmethionine: delta 24-sterol-C-methyl transferase (EC2.1.1.41) which normally produces cholesta-5,7,24-trienol and cholesta-5,7,22,24-tetraenol as the major sterols (total 4,4-desmethyl sterol content-8.3 fg/cell) was shown to synthesize trace levels of cholesterol (0.08 fg/cell). The identity of cholesterol was established by co-chromatography in TLC, GLC and HPLC with an authentic sample, mass spectroscopy and after an incubation with [1-14C]acetate by isotopic dilution and recrystallization of the radiochemically purified material to constant specific activity.