The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. The first step in the pathway is the condensation of acetyl-CoA and alpha-ketoglutarate into homocitrate, and this step is carried out by the enzyme homocitrate synthase (EC 4.1.3.21). In spite of extensive genetic analysis, no mutation affecting this step has been isolated until now in model organisms such as Saccharomyces cerevisiae or Neurospora crassa, although identification of mutations affecting the structural gene (LYS1) for homocitrate synthase was reported in the yeast Yarrowia lipolytica several years ago. Here we used these mutants for the cloning and sequencing of the Yarrowia LYS1 gene. The LYS1 gene encodes a predicted 446 amino acid polypeptide, with a molecular mass of 48442 Da. The Lys1p sequence displays two regions, one near the N-terminal section and the other in the central region, that contain conserved signatures found in prokaryotic homocitrate synthases (nifV genes of Azotobacter vinelandii and Klebsiella pneumoniae), as well as in all alpha-isopropyl malate synthases so far described. A putative mitochondrial targeting signal of 41-45 amino acids is predicted at the N-terminus. The Lys1p sequence shows 84% identity at the amino acid level with the putative product of open reading frame D1298 of S. cerevisiae. Northern blot hybridizations revealed a LYS1 transcript of approximately 1.7 kb in Y. lipolytica. Deletion of the LYS1 gene resulted in a Lys- phenotype. Our results indicate that we cloned the structural gene for homocitrate synthase in Y. lipolytica, and that the enzyme is encoded by a single gene in this yeast.