Two recessive alleles of ERG10 and three temperature-sensitive recessive alleles of HMG1 (3-hydroxy-3-methyl-glutaryl-CoA reductase isoenzyme 1) were isolated in a screen for mevalonate auxotrophs in Saccharomyces cerevisiae. The essential, single-copy ERG10 gene was cloned by complementation of the temperature-sensitive phenotype of erg10-21. The 1,194-base pair continuous open reading frame, encoding a 398-amino acid polypeptide with a calculated molecular mass of 41,681 daltons, was demonstrated to encode cytoplasmic aceto-acetyl-CoA thiolase. Acetoacetyl-CoA thiolase activity corresponded to the number of copies of ERG10 present in cell extracts, and null alleles of ERG10 produced no detectable acetoacetyl-CoA thiolase enzyme activity. The deduced amino acid sequence was 40-95% identical to acetoacetyl-CoA thiolases from other organisms. This identity included the active site cysteines located at amino acids 91 and 384 in the Erg10 protein.