One major unresolved issue in muscle blood flow regulation is that of the role of circulating versus interstitial vasodilatory compounds. The present study determined adenosine-induced formation of NO and prostacyclin in the human muscle interstitium versus in femoral venous plasma to elucidate the interaction and importance of these vasodilators in the 2 compartments. To this end, we performed experiments on humans using microdialysis technique in skeletal muscle tissue, as well as the femoral vein, combined with experiments on cultures of microvascular endothelial versus skeletal muscle cells. In young healthy humans, microdialysate was collected at rest, during arterial infusion of adenosine, and during interstitial infusion of adenosine through microdialysis probes inserted into musculus vastus lateralis. Muscle interstitial NO and prostacyclin increased with arterial and interstitial infusion of adenosine. The addition of adenosine to skeletal muscle cells increased NO formation (fluorochrome 4-amino-5-methylamino-2',7-difluorescein fluorescence), whereas prostacyclin levels remained unchanged. The addition of adenosine to microvascular endothelial cells induced an increase in NO and prostacyclin levels. These findings provide novel insight into the role of adenosine in skeletal muscle blood flow regulation and vascular function by revealing that both interstitial and plasma adenosine have a stimulatory effect on NO and prostacyclin formation. In addition, both skeletal muscle and microvascular endothelial cells are potential mediators of adenosine-induced formation of NO in vivo, whereas only endothelial cells appear to play a role in adenosine-induced formation of prostacyclin.