The present study demonstrates that dexamethasone and 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (CPT-cAMP), a cAMP analog, increase the substrate flux through branched-chain keto acid dehydrogenase (BCKDH) in primary rat hepatocytes cultured in defined medium. Maximum response (2.7-fold increase in flux) was observed when hepatocytes were cultured with 1 microM dexamethasone plus 50 microM CPT-cAMP for 24 h. This increase in the flux rate was accompanied by significant increases in both the basal and total activities of BCKDH (2.2- and 2.0-fold, respectively), without any significant change in the activity state of this enzyme. The increase in BCKDH activity was the result of increased protein mass of E1 alpha (3.2-fold), E1 beta (2.9-fold), and E2 (1.6-fold) subunits of BCKDH, indicating that E2 is the limiting subunit for the expression of BCKDH. The relative abundance of mRNAs encoding the E1 alpha, E1 beta, and E2 subunits of BCKDH increased by 7.4-, 21.7-, and 4.8-fold, respectively. We conclude that increased flux through BCKDH in hepatocytes cultured with dexamethasone and CPT-cAMP is due to increased expression of BCKDH subunit genes. However, nonstoichiometric expression of individual subunits and the corresponding mRNAs suggests regulation of BCKDH also at translational and post-translational steps.