Two sites per subunit of the dihydrolipoyl transacetylase component of the kidney pyruvate dehydrogenase complex can be acetylated, and high levels of acetylation can be achieved under conditions in which only a few subunits of the pyruvate dehydrogenase component are functional (Cate, R. L., and Roche, T. E. (1979) J. Biol. Chem 254, 1659-1665). These obsewations suggest intersite transfer of acetyl groups between lipoyl moieties (two per subunit) of the dihydrolipoyl transacetylase component. We have used flowquench rapid reaction studies to evaluate whether a relay system functions to transfer acetyl groups rapidly between sites on the dihydrolipoyl transacetylase core. In complexes containing reduced levels of active pyruvate dehydrogenase component, four to seven acetyl groups per active pyruvate dehydrogenase component are introduced at a constant rate nearly equal, for the fraction of active enzyme, to the rate of the overall reaction catalyzed by the complex. The initial round of decarboxylation proceeds at a much faster rate than the incorporation of acetyl groups. Since both the first and second round of acetylation occurs at a constant rate equal to the rate of the overall reaction, reductive acetylation is the rate-limiting step in the overall reaction. Furthermore, if only one lipoyl moiety services an a/3 unit of the pyruvate dehydrogenase component, the above data indicate that there is subsequent interlipoyl transfer of acetyl groups which occurs at a faster rate than the reductive acetylation reaction.