Carbohydrate depletion precipitates fatigue in skeletal muscle but, because pyruvate provides both acetyl-CoA for mainline oxidation and anaplerotic carbon to the citric acid cycle (CAC), the mechanism remains obscure. Thus, pyruvate and CAC kinetic parameters were independently quantified in mitochondria isolated from rat mixed skeletal muscle. Mitochondrial oxygen consumption rate (J_o) was measured polarographically while either pyruvate or malate was added stepwise in the presence of a saturating concentration of the other substrate. These substrate titrations were carried out across a physiological range of fixed extramitochondrial ATP free energy states (G_P), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent K_M-malate for mitochondrial J_o ranged from 21 to 32 µM, and the apparent K_M-pyruvate ranged from 12 to 26 µM, with both substrate K_M values increasing as G_P declined. The V_MAX for both substrates also increased as G_P fell, reflecting thermodynamic control of J_o. Reported in vivo skeletal muscle malate concentrations are over tenfold greater than the K_M-malate determined in this study. In marked contrast, the K_M-pyruvate determined here is near the [pyruvate] reported in muscle approaching exhaustion associated with glycogen depletion. When the data were evaluated in the context of a linear thermodynamic force:flow (G_P:J_o) relationship, the G_P:J_o slope was essentially insensitive to changes in [malate] in the range observed in vivo, but decreased markedly with declining [pyruvate] across the physiological range. Mitochondrial respiration is particularly sensitive to variations in [pyruvate] in the physiological range. In contrast, physiological [malate] exerts very little, if any, influence on mitochondrial pyruvate oxidation measured in vitro.