The cdc60 mutation (for cell division cycle) of the yeast, Saccharomyces cerevisiae, confers arrest at the START point of the cell cycle upon shift to the restrictive temperature [Bedard et al., Curr. Genet. 4 (1981) 205-214]. We have cloned the CDC60 gene by complementation of the temperature-sensitive phenotype. Sequence analysis revealed a single open reading frame of 3270 bp and the deduced amino acid sequence showed 50.5% sequence identity to the cytosolic leucyl-tRNA synthetase (LeuRS) from Neurospora crassa, implying that CDC60 encodes the corresponding yeast protein. Thus, CDC60 does not appear to be involved directly in the regulation of the cell cycle. Rather, the cdc60 mutation leads to cell-cycle arrest at the nutrient control point START due to a deficiency of charged leucyl-tRNA. The CDC60 gene product also shows homology to LeuRSs from other organisms and to aminoacyl-RS for isoleucine, valine and methionine.