To investigate the mechanism of the carboxylation of pyruvate to oxalacetate catalyzed by the enzyme transcarboxylase, we have measured the D(V/K) and 13(V/K) isotope effects. Comparison of the double-reciprocal plots of the initial velocities with [1H3]pyruvate and with [2H3]pyruvate as substrate yields a deuterium isotope effect on Vmax/Km of 1.39 +/- 0.04. The 13C kinetic isotope effect on the carboxylation of pyruvate to oxalacetate has been measured by the competitive method and is 1.0227 +/- 0.0008. To determine whether the removal of the proton from pyruvate and the addition of the carboxyl group occur in the same or in different steps, the double-isotope fractionation test has been used. When [2H3]pyruvate replaces [1H3]pyruvate as the substrate, the observed 13(V/K) isotope effect falls from 1.0227 to 1.0141 +/- 0.001. The latter value is in excellent agreement with the value of 1.0136, predicted for a stepwise pathway. We may conclude, therefore, that the carboxylation of pyruvate catalyzed by transcarboxylase proceeds by a stepwise mechanism involving the intermediate formation of the substrate carbanion.