AbstractA DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl‐coenzyme A (acetyl‐CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromsome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79·2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons dowunstream, is in a more conventional context. Possible implications of two alternative traslational start sites for the celular lacalization of the enzyme are disussed. A stable mutant of this gene, obtained by the gene disruptiona technique, had the same low basal acitivity of acetyl‐CoA synthetase as wild‐type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetlyl‐CoA synthetase (ACSI) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction os isocitrate lyase on acetate media, indicating that activity of acetyl‐CoA sytheatase is dispensable for incuctionof the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACSI gene did not affect growth on media containing ethanol as the sole crbon source. Demonstrating that threre are alternative pathways pathways leading to acetyl‐CoA under these condintions.