The activity and turnover of dihydrolipoamide dehydrogenase (E3), the common component of the three 2-oxoacid dehydrogenase complexes, were measured during the differentiation of 3T3-L1 preadipocytes into 3T3-L1 adipocytes. The specific activity of E3 increased approx. 3-4-fold in 3T3-L1 adipocytes differentiated under a regimen of insulin, dexamethasone and 3-isobutyl-1-methylxanthine for 48 h, followed by insulin alone thereafter. A rabbit antibody to pig heart E3 quantitatively precipitated the enzyme from 3T3-L1 adipocytes. By using immunoprecipitation and gel electrophoresis, a 3.3-fold increase was observed in E3 protein in 3T3-L1 adipocytes as compared with 3T3-L1 preadipocytes, on a DNA basis. Pulse-labelling experiments with L-[35S]methionine revealed a 3.5-fold increase in the rate of synthesis of E3 in 3T3-L1 adipocytes compared with that observed in 3T3-L1 preadipocytes. In contrast, the apparent half-lives of the E3 in 3T3-L1 preadipocytes (43 h) and 3T3-L1 adipocytes (33 h) were not significantly different. Therefore, the 3-4-fold increase in the specific activity of E3 in 3T3-L1 adipocytes resulted from an increased rate of synthesis of the enzyme.