Acetyl‐CoA hydrolase, which hydrolyzes acetyl‐CoA to acetate and CoASH, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2‐terminally blocked. The enzyme was purified 1080‐fold to apparent homogeneity by successive purification steps using DEAE‐Sepharose, gel filtration and hydroxylapatite. The molecular mass of the native yeast acetyl‐CoA hydrolase was estimated to be 64 ± 5 kDa by gel‐filtration chromatography. SDS/PAGE analysis revealed that the denatured molecular mass was 65 ± 2 kDa and together with that for the native enzyme indicates that yeast acetyl‐CoA hydrolase was monomeric. The enzyme had a pH optimum near 8.0 and its pI was approximately 5.8. Several acyl‐CoA derivatives of varying chain length were tested as substrates for yeast acetyl‐CoA hydrolase. Although acetyl‐CoA hydrolase was relatively specific for acetyl‐CoA, longer acyl‐chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl‐CoA. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine‐modifying reagent.