An enzyme that can synthesize O-alkylhomoserine from alcohols and O-acetylhomoserine was purified from Corynebacterium acetophilum. The enzyme was found to be identical to O-acetylhomoserine sulfhydrylase; a preparation that appeared homogeneous on polyacrylamide gel electrophoresis showed both O-alkylhomoserine-synthesizing and O-acetylhomoserine sulfhydrylase activities. Its molecular weight was determined to be about 220,000, and it consisted of two subunits. Its pH and temperature optima for the two reactions were the same. Besides catalyzing the formation of homocysteine from O-acetylhomoserine and sulfide, it also catalyzed the syntheses of O-alkylhomoserines corresponding to the alcohols added form O-acetylhomoserine and ethyl alcohol, n-propylalcohol, n-butyl alcohol, methyl alcohol, and n-pentyl alcohol, its activities with these alcohols decreasing in that order. L-Homoserine, O-succinylhomoserine, and O-acetylserine reacted with sulfide. O-ethylhomoserine, O-acetylthreonine, O-succinylhomoserine, and O-acetylserine inhibited both enzyme activities. O-acetylhomoserine sulfhydrylase purified from Saccharomyces cerevisiae also showed O-alkylhomoserine-synthesizing activity. Thus, O-acetylhomoserine sulfhydrylase seems to catalyze O-alkylhomoserine synthesis in the presence of appropriate concentrations of alcohol and O-acetylhomoserine in microorganisms.