The metabolism of piromidic acid (PA, 5, 8-dihydro-8-ethyl-5-oxo-2-pyrrolidinopyrido [2, 3-d] pyrimidine-6-carboxylic acid=pyrrolidino-PPA) by rat liver preparations was studied. The first step of PA metabolism was found to be hydroxylation at the 2- or 3-position in the pyrrolidine ring by the mixed-function oxidase system in liver microsomes to form M-II (2-hydroxypyrrolidino-PPA) or M-V (3-hydroxypyrrolidino-PPA). M-V did not undergo successive oxidation with hepatic 105000×g supernatant and microsomal preparations, whereas M-II was further metabolized not only by the hepatic 105000×g supernatant, to form the corresponding γ-aminobutyric acid derivative (RNHCH₂CH₂CH₂-COOH, M-IV), but also converted by the hepatic microsomes to 2, 5-dihydroxypyrrolidine derivative (M-VI), which was in turn converted to form amino derivative (M-III, amino-PPA). This conversion is highly likely to be non-enzymatic degradation. Glucuronicacid conjugate of PA was found to be produced from PA with hepatic 9000×g supernatant preparations containing uridine-5'-diphosphoglucuronic acid and D-saccharic acid-1, 4-lactone. Any evidences implying extrahepatic biotransformation by the blood or kidney preparation were not found of PA and its metabolites except M-II which was found to be converted to M-IV in both incubation media.