Glutamate dehydrogenase (NAD) activity was measured in liver and diaphragm mitochondria from 48 h fasted rats. Kinetic studies were performed with diaphragm enzyme and the effects of L-leu, ADP and L-ala on the K(1)m and V(1)max for NH(4)+, and α-ketoglutarate were evaluated. L-leucine increases by 2-8 fold the V(1)max for all three substrates with no significant changes in the K(1)m. ADP increased by 3-7 fold the V(1)max for all three substrates and the K(1)m for NADH and α-ketoglutarate by 1.5-7.0 fold. L-alanine had no effect on either the V(1)max or the K(1)m of any substrate. The results suggest that muscle has the capacity to form glutamate through the glutamate dehydrogenase reaction and that L-leucine may stimulate this reaction in muscle.