A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as Alcaligenes faecalis. The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of L-pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg²⁺ and K⁺. The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction, K_eq = [L-glutamate]/[L-pyroglutamate], was evaluated to be approximately 0.035, which indicates that the reaction tends to form L-pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.