We have cloned, sequenced and physically mapped the CYS3 gene of Saccharomyces cerevisiae. This gene can complement the cys3-1 allele, and disruptions at this locus lead to cysteine auxotrophy. The predicted CYS3 product is closely related (46% identical) to the rat cystathionine gamma-lyase (Erickson et al., 1990), but differs in lacking cysteine residues. These results provide further evidence that the S288C strain of yeast resembles mammals in synthesizing cysteine solely via a trans-sulfuration pathway. The CYS3 product was found to have strong homology to three other enzymes involved in cysteine metabolism: the Escherichia coli metB and metC products and the S. cerevisiae MET25 gene product. The trans-sulfuration enzymes appear to form a diverged family and carry out related functions from bacteria to mammals.