2,3-Oxidosqualene (OS) cyclase (OSC) catalyzes the conversion of OS to lanosterol, an essential step in the biosynthesis of sterols. The Candida albicans gene (ERG7) encoding OSC was cloned by complementation of a Saccharomyces cerevisiae OSC mutant (erg7). Two different Erg+ clones were isolated that contain a common overlapping region. The minimum region required for complementation was determined to be approx. 3.2 kb and a single 2.7-kb ERG7 transcript was detected. The cloned Candida ERG7 DNA complemented an additional nonconditional erg7 allele and a temperature-sensitive erg7 mutation. OSC activity was restored in the mutants as determined by [14C]acetate incorporation in vivo as well as incorporation in vitro in cell-free extracts using either [14C]isopentenyl pyrophosphate or [3H]OS as substrate. The level of OSC produced from expression of a single copy of the Candida ERG7 sequence was sufficient to allow growth of the S. cerevisiae erg7 mutants in the absence of exogenous ergosterol. These data support the contention that the Candida ERG7 sequence is the structural gene for OSC.