1. We investigated the effect of moderate systemic hypoxia on the arterial, venous and interstitial concentration of adenosine and adenine nucleotides in the neurally and vascularly isolated, constant-flow perfused gracilis muscles of anaesthetized dogs. 2. Systemic hypoxia reduced arterial PO2 from 129 to 28 mmHg, venous PO2 from 63 to 23 mmHg, arterial pH from 7.43 to 7.36 and venous pH from 7.38 to 7.32. Neither arterial nor venous PCO2 were changed. Arterial perfusion pressure remained at 109 +/- 8 mmHg for the first 5 min of hypoxia, then increased to 131 +/- 11 mmHg by 9 min, and then decreased again throughout the rest of the hypoxic period. 3. Arterial adenosine (427 +/- 98 nM) did not change during hypoxia, but venous adenosine increased from 350 +/- 52 to 518 +/- 107 nM. Interstitial adenosine concentration did not increase (339 +/- 154 nM in normoxia and 262 +/- 97 nM in hypoxia). Neither arterial nor venous nor interstitial concentrations of adenine nucleotides changed significantly in hypoxia. 4. Interstitial adenosine, AMP, ADP and ATP increased from 194 +/- 40, 351 +/- 19, 52 +/- 7 and 113 +/- 36 to 764 +/- 140, 793 +/- 119, 403 +/- 67 and 574 +/- 122 nM, respectively, during 2 Hz muscle contractions. 5. Adenosine, AMP, ADP and ATP infused into the arterial blood did not elevate the interstitial concentration until the arterial concentration exceeded 10 microM. 6. We conclude that the increased adenosine in skeletal muscle during systemic hypoxia is formed by the vascular tissue or the blood cells, and that adenosine is formed intracellularly by these tissues. On the other hand, adenosine formation takes place extracellularly in the interstitial space during muscle contractions.