During contraction of skeletal muscle in the isolated dog hindlimb under constant-flow perfusion, the specific activity of infused [8- 14C]adenosine (nonvasoactive concn) in venous effluents decreased to 7% of the resting level in 1.25 min and was associated with a concomitant decrease in vascular resistance to 39% of the resting value. Since this decrease in specific activity of labeled adenosine could have been due to 1) an enhanced adenosine release by parenchymal tissue, 2) an exercise-induced increase in the number of open capillaries (greater surface area) in the absence of increased adenosine production 3) some degree of tissue hypoxia, or 4) a combination of these factors, experiments with maximally dilated vessels were performed. Acetylcholine and a nonvasoactive concentration of [8- 14C]adenosine were continuously infused into an isolated dog hindlimb which was perfused at constant flow during periods of rest, contraction, and recovery while arterial oxygenation was maintained at normoxic levels. Approximately 2.75 min after the onset of contraction with the vascular bed maximally dilated, the specific activity of [8- 14C]adenosine in venous effluents decreased to 38% of the resting level while the venous Po2 decreased from 78 to 42 mmHg; the value of 42 mmHg indicates apparent absence of hypoxia in the muscle. These observations are consistent with the concept that adenosine release is involved in the vasodilation observed in contraction of skeletal muscle.