A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%). The N-terminal half of it was homologous (39%) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYI1) gene marginally complemented the E. coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E. coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine gamma-lyase and E. coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.