[(S)-16O,17O]Thiophosphoenolpyruvate has been used as a substrate in H218O for the reaction catalyzed by phosphoenolpyruvate carboxylase, and the absolute configuration of the product, inorganic [16O,17O,18O]thiophosphate, has been determined. The reaction proceeds with inversion of configuration at phosphorus, thus ruling out the cyclic mechanism that has been proposed for this enzyme. The stereochemical result is consistent with a stepwise mechanism involving the intermediate formation of carboxyphosphate.