Tryptophan synthase of Saccharomyces cerevisiae has been purified from a derepressed mutant to homogeneity. The crucial step in the new procedure is hydrophobic interaction chromatography on pentyl‐Sepharose in strong ammonium sulfate solutions. Tryptophan‐synthase‐inactivating agents are efficiently removed by this step. The protein migrates as a single boundary in the ultracentrifuge with a sedimentation coefficient of 6.1 S. Its molecular weight, as judged by sedimentation equilibrium measurements, equals 167000. Treatment of the enzyme with sodium dodecylsulfate followed by gel electrophoresis in polyacrylamide reveals a single band with an apparent molecular weight of 76000. Gel filtration studies with Sephacryl S‐300, performed under conditions that suppress unspecific absorption of proteins, indicate a molecular weight of 390000. Tryptophan synthase from Escherichia coli, having a molecular weight of 148000, is also eluted at a position corresponding to an apparently higher molecular weight of 280000. These results are interpreted in the context of other genetic and biochemical information on tryptophan synthase from S. cerevisiae, Neurospora crassa, and E. coli as signifying that the yeast enzyme is a dimer of bifunctional polypeptide chains. Previously, tryptophan synthase from S. cerevisiae was regarded as an oligomer of four polypeptide chains of molecular weight 37000 each. It is probably the product of inadvertent proteolysis.