Despite genes of the sterol methyl-oxidase component (SMO) of the sterol-C4-demethylation multienzymatic complex have been identified in a variety of organisms and the key role played by SMO in yeast sterol biosynthesis, the enzymological properties of yeast SMO have not been investigated. An enzymatic assay for measuring specifically sterol 4α-methyl-oxidase activity in Saccharomyces cerevisiae has been developed for the first time by using [¹⁴C]-4,4-dimethyl-zymosterol as substrate. It allowed enzymatically formed C4 mono- and di-demethylated products to be characterized as well as two novel C4-hydroxymethyl-zymosterol derivatives to be identified as immediate oxidative metabolites by the yeast 4,4-dimethyl-zymosterol 4α-methyl-oxidase (ScSMO). The properties of microsomal ScSMO have been established with respect to cofactor requirements and kinetics and the substrate selectivity examined with a number of 4,4-dimethyl- and 4α-methyl-sterols. Remarkably, ScSMO showed very low activity with 24-methylene-24-dihydrocycloartenol, the natural substrate of maize 4,4-dimethyl-sterol-C4-methyl-oxidase. Conversely, maize sterol-C4-methyl-oxidases showed extremely reduced activity with the natural substrate of ScSMO. The previously described antifungal agent, 6-amino-2-n-pentylbenzothiazole was shown to directly inhibit the microsomal ScSMO activity in vitro. The yeast system was more than 500 times more sensitive to this derivative than the maize systems. These distinct substrate specificities and inhibitor sensitivities between yeast and plant sterol-4α-methyl-oxidases probably reflect diversity in the structure of their active sites in relation to the distinct sterol biosynthetic pathways.