Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating the intracellular acetyl-CoA or CoASH pools. The yeast enzyme is encoded by ACH1 (acetyl-CoA hydrolase) and the expression of ACH1 is repressed by glucose (Lee, F.-J.S., Lin, L.-W. and Smith, J.A. (1990) J. Biol. Chem. 265, 7413-7418). In order to study the biological function of the acetyl-CoA hydrolase, a null mutation (achl-1) was created by gene replacement. The mutation, while not lethal, slows down acetate utilization. In comparison to wild-type, homozygote ach1-1 diploids, the onset of sporulation was delayed. When measuring the levels of ACH1 mRNA and acetyl-CoA hydrolase activity, we demonstrated that ACH1 was highly expressed during sporulation process. These results indicated that acetyl-CoA hydrolase in yeast cells involved in acetate utilization and subsequently affected the sporulation process.