Maximal rates, coupling, and control of oxidative phosphorylation were studied in isolated skeletal muscle mitochondria from rat and rabbit. Mitochondria were incubated under various conditions of temperature, pH, and substrate availability. A 20% decrease in coupling (ADP/O) was observed at 43°C as compared to 37°C in rat mixed skeletal muscle mitochondria. Changes in pH from 7.00 to 6.20 affected neither coupling nor maximal (state 3) respiration rates. Changing the substrate supply from pyruvate to palmitoyl-carnitine (+ malate) did not alter ADP/O, but markedly degraded the energy state sustained at submaximal ATP turnover. Thus, carbohydrate depletion may be associated with inhibition of contractile function and the recruitment of less economical higher threshold motor units. State 3 respiration of mitochondria from rabbit Type IIb fibers oxidizing pyruvate + malate + α-glycerophosphate was 27% higher than that of mitochondria from Type I rabbit skeletal muscle. However, the ADP/O ratio in the Type IIb preparation was 18% lower. The experimental findings suggest that temperature, substrate supply, and energetic differences between slow twitch and fast twitch motor units may impact the economy of mitochondrial oxygen utilization during heavy aerobic exercise, and thus contribute to the slow component of oxygen uptake.