An undialyzable, thermostable inhibitor of tryptophan synthase from pea plants, Escherichia coli, and Neurospora crassa occurs in the filtrate from acetone extraction of pea tissue. A procedure is described for purification of the inhibitor extracted from roots. The final, purified preparation is homogeneous as shown by disc electrophoresis on acrylamide gel. The molecular weight, estimated by gel filtration on Sephadex G-200, is about 18 000. The inhibitor is stable even on heating at pH 7.0 in a water bath at 100 °C and has a characteristic ultraviolet absorption spectrum. The inhibitor shows some protein-like properties and acid hydrolysates were found to contain substances sensitive to ninhydrin. However, the hydrolysates also contain substances, presumably sugars, sensitive to benzidine reagent, appreciable sugar in galactose equivalents, and some fluorescent substances. The inhibitor was also found in shoot tissue. In both root and shoot the inhibitor increases, on a protein basis, with age of the tissue.