AbstractPurification of Saccharomyces cerevisiae cystathionine γ‐lyase (γ‐CTLase) was hampered by the presence of a protein migrating very close to it in various types of column chromatography. The enzyme and the contaminant were nevertheless separated by polyacrylamide gel electrophoresis. N‐terminal amino acid sequence analysis indicated that they are coded for by CYS3(CYI1) and MET17(MET25), respectively, leading to the conclusion that CYS3 is the structural gene for γ‐CTLase and that the contaminant is O‐acetylserine/O‐acetylhomoserine sulfhydrylase (OAS/OAH SHLase). Based on these findings, we purified γ‐CTLase by the following strategy: (1) extraction of OAS/OAH SHLase from a CYS3‐disrupted strain; (2) preparation of antiserum against it; (3) identification of a strain devoid of the OAS/OAH SHLase protein using this antiserum; and (4) extraction of γ‐CTLase from this strain. Purified γ‐CTLase had cystathionine γ‐synthase (γ‐CTSase) activity if O‐succinylhomoserine, but not O‐acetylhomoserine, was used as substrate. From this notion we discuss the evolutional relationship between S. cerevisiae γ‐CTLase and Escherichia coli γ‐CTSase.