In muscles of rats with metabolic acidosis, branched-chain alpha-ketoacid dehydrogenase (BCKAD) activity is increased but the signal initiating this response is unknown. Potential signals include intracellular responses to acidemia and/or glucocorticoids. It is not known whether the signal activates BCKAD by changing the proportion of inactive (phosphorylated) or active (dephosphorylated) enzyme or increases the amount of enzyme. To separate the effects of extracellular pH and glucocorticoids on BCKAD, enzyme activity was measured in two cell types: (1) LLC-PK1 cells that do not express glucocorticoid receptors and (2) LLC-PK1-GR101 cells that express rat glucocorticoid receptors. Extracellular acidification (pH 6.95) increased the maximal BCKAD activity, the percentage of active enzyme, and the amounts of BCKAD protein. Dexamethasone also increased the percentage of active BCKAD but the time required for activation was longer than with acidification. These results demonstrate that BCKAD is dually regulated by extracellular pH and glucocorticoids.