The nutritional requirements of three yeast mutants, previously shown to possess low O-acetyl-L-serine (OAS) and O-acetyl-L-homoserine (OAH) sulfhydrylase activities, were reinvestigated. It was thus found that one mutant (strain No. 16), previously identified as a homocysteine auxotroph, is in fact a double mutant requiring both cysteine and OAH. In agreement with the previous assignment, the other two strains (strains No. 13 and 17) were shown to be true cysteine auxotrophs. These results can best be explained by assuming the cystathionine pathway to be the main route of homocysteine synthesis in this organism. It was further found that extracts of the three mutants contain genetically modified OAS-OAH sulfhydrylases with much reduced catalytic activities. Modified sulfhydrylase was partially purified from strain No. 16 by the same procedure as for the wild-type enzyme. Both OAS and OAH sulfhydrylase activities of the mutant enzyme were copurified and behaved identically on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the purified mutant enzyme were shown to be very similar to those of the wild-type enzyme, except that the catalytic activities of the former were only 3-5% of those of the latter, and that the ratio of OAH sulfhydrylase to OAS sulfhydrylase activity was somewhat lower in the former than in the latter.