The influence of systemic hypoxia on lactate and adenosine output from isolated constant-flow-perfused gracilis muscle was determined in anesthetized dogs. The lactate transport inhibitor alpha-cyano-4-hydroxycinnamic acid (CHCA) was employed to distinguish the direct effects of hypoxia on adenosine output from the effects produced indirectly by a change in lactate concentration. Reduction of arterial PO2 from 135 +/- 4 to 39 +/- 2 mmHg raised arterial lactate from 1.26 +/- 0.32 to 2.22 +/- 0.45 mM but decreased venoarterial lactate difference from 0.53 +/- 0.09 to -0.13 +/- 0.19 mM, indicating that lactate output from the muscle was abolished. Arterial adenosine did not change, but venoarterial adenosine difference increased from 20.6 +/- 10.1 to 76.5 +/- 14.4 nM. CHCA infusion during hypoxia abolished adenosine output from gracilis muscle (venoarterial adenosine difference = -20.5 +/- 40.6 nM). In isolated rat soleus muscle fibers, intracellular pH increased from 6.96 +/- 0.04 to 7.71 +/- 0.14 in response to a reduction of PO2 from 459 +/- 28 to 53 +/- 3 mmHg. Correspondingly, adenosine output decreased from 3.71 +/- 0.15 to 3.04 +/- 0.27 nM. These data suggest that hypoxia did not directly stimulate adenosine output from red oxidative skeletal muscle, but rather systemic hypoxia increased lactate delivery and the resulting increase in intracellular lactate decreased intracellular pH, which stimulated adenosine output.