Abstract Phenylalanine ammonia-lyase from the yeast Rhodotorula glutinis was purified by salt fractionations and Sephadex chromatography. Density gradient centrifugation and Sephadex chromatography indicated its molecular weight to be about 275,000. Enzymatic deamination of several ring-substituted phenylalanine analogues and d-phenylalanine was studied. While cinnamic acid, a product of deamination, acted as a competitive inhibitor, phenylpropiolic acid, its acetylenic analogue, was a noncompetitive inhibitor. This latter finding is consistent with the presence of more than one form of enzyme during the reaction sequence. The enzyme was inactivated by borohydride or cyanide. In addition, 0.7 to 1.2 atoms of tritium (incorporated from NaB3H4) or about 1.4 moles of cyanide completely inactivated 1 mole of enzyme. After acid hydrolysis of the enzyme labeled with Na14CN, the radioactivity was recovered primarily as 14C-aspartic acid. The 4-carboxyl group of this aspartate was radioactive. Hydrolysis of 3H-NaBH4-inactivated enzyme yielded radioactive alanine. This evidence indicates that bound dehydroalanine is at the active site of the enzyme.