1. Maximal calcium-activated force (Fmax) and calcium sensitivity were markedly decreased in detergent-skinned fibres from skeletal and cardiac muscle by solutions that mimicked the total milieu changes associated with fatigue and hypoxia. Further experiments determined the relative contribution of each of the individual changes in milieu. 2. Both Ca2+ sensitivity and Fmax of skeletal and cardiac fibres were decreased with increased [H+] or inorganic phosphate (Pi). These effects were greater in cardiac muscle. 3. Decreasing MgATP over the range observed with fatigue and hypoxia (6.8-4.7 mM) had no effect on Fmax or Ca2+ sensitivity of either muscle type. 4. Decreasing phosphocreatine (PCr: 15-1 mM) increased Fmax but had little effect on Ca2+ sensitivity in both muscle types. In cardiac fibres, the effect on Fmax could be mimicked by inhibition of endogenous creatine kinase. 5. ADP (0.7 mM) increased Fmax and Ca2+ sensitivity, while AMP (0.06 mM) slightly increased Fmax but had no effect on Ca2+ sensitivity of either skeletal or cardiac fibres. 6. Creatine (25 mM) had no significant effect on either Ca2+ sensitivity or Fmax of skeletal and cardiac muscle fibres. At higher levels (50 mM), however, creatine depressed Fmax and slightly altered Ca2+ sensitivity. 7. Thiophosphorylation of myosin P light chains (phosphorylatable light chains of myosin) in rabbit psoas fibres had no effect on Ca2+ sensitivity, yet slightly but significantly increased Fmax under fatigue conditions. 8. Reducing the affinity for ATP hydrolysis (by adding ADP, AMP and creatine) over the range calculated for fatigue/hypoxia (60-45 kJ/mol) produced the enhancement in Fmax expected from added ADP and AMP in cardiac but not skeletal muscle, indicating that changes in affinity influence Fmax of skeletal muscle. Reducing affinity produced little change in Ca2+ sensitivity of skeletal muscle. In contrast, the change produced in cardiac muscle was greater than that expected from addition of ADP and AMP; i.e. decreasing affinity increases calcium sensitivity of the heart. 9. Simple summation of all significant changes expected from each constituent altered by fatigue/hypoxia adequately predicted the observed changes in Fmax and Ca2+ sensitivity in both cardiac and skeletal muscle fibres with but one exception (the change in Ca2+ sensitivity of skeletal muscle at pH 7 was slightly overestimated).