Until recently, little evidence existed for the stepwise breakdown by mammalian enzymes of hydroxyproline, an important constituent of total animal protein. In past years, several investigators (4-6) have determined the labeled amino acids formed when nn-2-C14-hydroxyproline is administered to rats. The earliest studies performed in vitro, however, indicated that Alpyrroline-3-hydroxy-5-carboxylate, y-hydroxyglutamic semialdehyde, and y-hydroxyglutamate might arise as intermediary metabolites (7,8). A later investigation (6) reported the formation of labeled y-hydroxyglutamate from n-‘2-C14-hydroxyproline by rat liver slices. These findings were unified when Adams and Goldstone (9, 10) defined the initial enzymatic steps in mammalian catabolism of hydroxyproline as, first, an oxidation of n-hydroxyproline to Al-pyrroline-3-hydroxy-5-carboxylate and, second, an oxidation of the latter compound to y-hydroxyglutamate. Several short papers appeared at approximately the same time reporting the presence of enzymes in rat liver extracts that metabolized y-hydroxyglutamate. The initial finding (1) that glyoxylate and alanine were formed enzymatically from this hydroxyamino acid was extended by the results of Kuratomi and Fukunaga (11). They reported the presence of an enzyme in rat liver extracts that catalyzed the condensation of glyoxylate and ppruvate to form cr-keto-y-hydroxyglutarate, which, in turn, was converted by transamination to y-hydroxyglutamate. Since this is a reversible process, the reactions also delineated a possible pathway for the conversion of y-hydroxyglutamate to glyoxylate plus pyruvate. Goldstone and Adams (12) substantiated these findings, indicating that y-hydroxyglutamate participated in a unique, metal-requiring transamination reaction. Examination of our enzyme system led us to propose a sequence of reactions (3), in which the net formation of glyoxylate and alanine from y-hydroxyglutamate occurred by a sequential loss of the amino group of the amino acid, followed by a cleavage and a reamination of one of the fragments, as illustrated in the following reactions.