Little information is available regarding the regulation of glutamate dehydrogenase in skeletal muscle. We investigated the regulation of glutamate dehydrogenase by branched-chain amino acids (BCAA) in skeletal muscles from rats and chicks and determined the effects of metabolic acidosis on the activity and regulation of this enzyme by BCAA in rat skeletal muscle. Skeletal muscle mitochondria were prepared from normal rats and chicks and acidotic rats. Mitochondrial glutamate dehydrogenase activity was measured in the presence or absence of BCAA. Metabolic acidosis was induced by feeding rats 1.5% NH4Cl as drinking water. Glutamate dehydrogenase activity was stimulated by leucine (P < 0.001) and isoleucine (P < 0.05) in rat muscles and by leucine (P < 0.05) in chick muscles in a concentration-dependent manner. Both leucine and isoleucine had their maximum effects at a concentration of 1 mM (45% by leucine and 27% by isoleucine in rat muscle; 36% by leucine in chick muscle). The maximum stimulatory effects of leucine and isoleucine in rat muscles were additive. Neither valine nor 2-oxoisocaproate had an effect on glutamate dehydrogenase activity in rat or chick muscles. In acidotic rats, the basal activity of skeletal muscle glutamate dehydrogenase was 1.8-fold (P < 0.01) greater than in control rats; leucine, isoleucine, and valine significantly increased glutamate dehydrogenase activity (maximally 86, 55 and 33%, respectively; P < 0.05). We conclude that glutamate dehydrogenase activity in skeletal muscle from rats and chicks is regulated by BCAA, and that a species difference exists between rats and chicks. Metabolic acidosis increases the activity of glutamate dehydrogenase and its sensitivity to BCAA.