5-Oxoprolinase catalyzes the ATP-dependent decyclization of 5-oxo-L-proline to L-glutamate. Previous studies provided evidence for the intermediate formation of a phosphorylated form of 5-oxoproline (Seddon, A. P., and Meister, A. (1986) J. Biol. Chem. 261, 11538-11541) and of a tetrahedral intermediate (Li, L., Seddon, A. P., and Meister, A. (1987) J. Biol. Chem. 262, 11020-11025). A new approach to the study of the reaction mechanism using the 18O isotope effect on the 13C NMR signals for 5-oxoproline and glutamate is reported here. The 13C chemical shifts induced by 18O substitution for the carbonyl group of 5-oxoproline and the gamma-carboxyl group of glutamate are about 0.03 ppm with respect to the corresponding 16O-compounds. Using 5-[18O]oxo[5-13C]proline (97 and 79.5 atom % excess, 13C and 18O, respectively), the disappearance of the 18O-labeled and unlabeled 5-oxoproline and formation of the corresponding glutamate species were followed in the reactions catalyzed by purified preparations of 5-oxoprolinase isolated from Pseudomonas putida and from rat kidney. This procedure permits simultaneous determinations of the rates of 18O exchange and of the overall decyclization reaction. The ratios of 18O exchange rates to the overall reaction rates for the bacterial and kidney enzyme catalyzed-reactions were 0.28 and 0.14, respectively. The findings support the view that the coupling of ATP hydrolysis to 5-oxoproline decyclization involves formation of a phosphorylated tetrahedal intermediate. Although the exchange phenomena are consistent with the mechanistic interpretations, they seem not to be required for catalysis.