The energetic consequences of strict oxyconformity in the intertidal worm S. nudus were studied by characterizing the PO2 dependence of respiration in mitochondria isolated from the body wall tissue. Mitochondrial respiration rose in a PO2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein-1 h-1. Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN. PO2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP). A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated PO2. Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria. The decrease in ATP/O ratios at higher PO2 was completely reversible. Addition of 90.9 μmol H2O2 l-1 did not inhibit ATP synthesis. Both observations suggest that oxidative injury did not contribute to oxyconformity. The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase. The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated PO2, at a slope significantly lower than the increment of state 3 respiration. Therefore, PO2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity. In conclusion PO2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria. The molecular nature of the alternative oxidase in lower invertebrates is still obscure. © 2001 Elsevier Science Inc.