The dihydrolipoyl transacetylase component (E2) of the pyruvate dehydrogenase complex catalyzes the reaction of acetyl coenzyme A (acetyl-CoA) with dihydrolipoamide, producing coenzyme A and 5-acetyldihydrolipoamide. The acetyl group is shown by experiments reported herein to be bonded to S8 in the enzymatic product. 1H NMR analysis of synthetic samples of both structural isomers of 5-acetyl-5-(phenylmercurio)dihydrolipoamide enabled structural assignments to be made. Reaction of 8-5-acetyl-6-5-(phenylmercurio)dihydrolipoamide with 3-mercaptopropionic acid in chloroform produced 8-5-acetyldihydrolipoamide which contained a small amount (5%) of the 6-S isomer. Reaction of 6,8-di-5-acetyldihydrolipoamide with NH2OH produced a 4:1 mixture of 6-5-acetyldihydrolipoamide and the 8-S isomer. These compounds did not isomerize at significant rates in chloroform but rapidly isomerized to the equilibrium mixture in aqueous solution (Keq= 3.4). The second-order rate constants for the hydroxide-catalyzed isomerization were found to be kf= (1.15 ± 0.07) X 106M−1.s−1 and kr = (3.36 ± 0.20) X 105M−1.s−1 in the direction of the formation of the 8-S isomer. The enzymatic product was trapped by addition of phenylmercuric hydroxide within 15 s-30 min after starting the reaction. 1H NMR analysis of the products obtained at various times showed that the enzymatic product was 8-S-acetyldihydrolipoamide, which underwent progressive isomerization to the mixture of isomers within a few minutes. In the reaction of acetyl-CoA with dihydrolipoamide, the latter substrate reacts in place of enzyme-bound dihydrolipoyl moieties. Therefore, acetylation occurs at the 8-S position of bound lipoyl groups.