Oxidative phosphorylation of isolated rat skeletal muscle mitochondria after exposure to lactic acidosis in either phosphorylating or nonphosphorylating states has been evaluated. Mitochondrial respiration and transmembrane potential (_m) were measured with pyruvate and malate as the substrates. The addition of lactic acid decreased the pH of the reaction medium from 7.5 to 6.4. When lactic acid was added to nonphosphorylating mitochondria, the subsequent maximal ADP-stimulated respiration decreased by 27% compared with that under control conditions (P < 0.05), and the apparent Michaelis-Menten constant (K_m) for ADP decreased to 10 µM vs. 20 µM (P < 0.05) in controls. In contrast, maximal respiration and ADP sensitivity were not affected when mitochondria were exposed to acidosis during active phosphorylation in state 3. Acidosis significantly increased mitochondrial oxygen consumption in state 4 (post-state 3), irrespective of when acidosis was induced. This effect of acidosis was attenuated in the presence of oligomycin. The addition of lactic acid during state 4 respiration decreased _m by 19%. The ratio between added ADP and consumed oxygen (P/O) was close to the theoretical value of 3 in all conditions. The addition of potassium lactate during state 3 (i.e., medium pH unchanged) had no effect on the parameters measured. It is concluded that lactic acidosis has different effects when induced on nonphosphorylating vs. actively phosphorylating mitochondria. On the basis of these results, we suggest that the influence of lactic acidosis on muscle aerobic energy production depends on the physiological conditions at the onset of acidity.