In Saccharomyces cerevisiae, the conversion of pyruvate to acetyl-coenzyme A may proceed directly via the pyruvate dehydrogenase complex (PDH) or indirectly via the so-called PDH bypass, which requires the sequential action of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-coenzyme A synthetase. The relative contribution of both pathways to the rate of acetyl-coenzyme A synthesis varies in an unknown way with cultural conditions. To determine the possible role of acetyl-coenzyme A synthetase in this central part of metabolism, we have analyzed the genes encoding this enzyme. Disruption of the recently cloned ACS1 gene [De Virgilio, C., Burckert, N., Barth, G., Neuhaus, J., Boller, T. & Wiemken, A. (1992) Yeast 8, 1043-1051] did not cause an apparent phenotype, except for a prolonged lag-phase during growth on glucose or C2 compounds such as acetate and ethanol. In fact, a product from a different gene is responsible for acetyl-coenzyme A formation in the acs1 mutant. We cloned a second gene encoding acetyl-coenzyme A synthetase, which we called ACS2. Inactivation of this gene caused inability to grow on media containing glucose, but not on media with acetate or ethanol as the sole carbon source. This indicates that ACS2 is essential for growth on glucose in batch cultures. The acs1-acs2 double mutant was not viable. The role of both genes in glucose metabolism and acetate or ethanol metabolism is discussed.