We tested the hypothesis that the respiratory function of skeletal muscle mitochondria is impaired by lactic acidosis and elevated concentrations of Pi. The rate of respiration of chemically skinned fiber bundles from rat soleus muscle was measured at [Pi] (brackets denote concentration) and pH values similar to those at rest (3 mM Pi, pH 7.0) and high-intensity exercise (20 mM Pi, pH 6.6). Respiration was measured in the absence of ADP and after sequential additions of 0.1 mM ADP, 20 mM creatine (Cr; VCr), and 4 mM ADP. Respiration at 0.1 mM ADP increased after addition of Cr. However, VCr was 23% lower (P < 0.05) during high-intensity conditions than during resting conditions. VCr was also reduced when Pi or H+ was increased separately (P < 0.05). Respiration in the absence of ADP and after additions of 0.1 mM ADP and 4 mM ADP was not affected by changes in [Pi] or [H+]. The response was similar, irrespective of when acidosis was induced (i.e., quiescent or actively respiring mitochondria). In conclusion, Cr-stimulated respiration is impaired by increases in [H+] and [Pi] corresponding to those in exercising muscle. Although the reduced Cr-stimulated respiration could be compensated for by increased [ADP], this might have implications for intracellular homeostasis.