Background: Previously, we have reported that local intra-arterial infusion of adenosine into cremaster muscle provided ischemic preconditioning effect against subsequent prolonged ischemia. In addition, local intra-arterial infusion of 8-sulfophenyl-theophylline (SPT; a nonspecific ADO receptor blocker) into cremaster muscle blocked microvascular protection induced by ischemic preconditioning. Purpose: To examine the role of adenosine in remote ischemic preconditioning (RIPC). Materials and Methods: Vascular pedicle isolated right cremaster model of the rat was used. Left femoral vessels were exposed and clamped for 45-60 minutes for RIPC (no clamped on sham RIPC group). To block the effects of RIPC, SPT (0.625mg/100g) was given through local intra-arterial infusion into cremaster muscle prior to RIPC. 4-hrs of warm ischemia in the right cremaster muscle was then applied and followed by 1-hr reperfusion. To mimic the effects of RIPC, adenosine (ADO; 350ug/kg/min) was given before the prolonged ischemia of cremaster muscle via intravenous infusion of femoral vein. The microcirculatory response in the cremaster muscle including terminal arteriole diameter, capillary perfusion and endothelial function (endothelium-dependent and -independent vasodilatation) was evaluated. Four groups (6 rats per group) were designed with 1) RIPC, 2) Sham RIPC, 3) SPT + RIPC, 4) ADO + Sham RIPC. Results: We found that 45-60mins ischemia in the left hindlimb of rat induced a significant microvascular protection in right cremaster muscle against later prolonged ischemia. After 4-hours of warm ischemia, the average diameter of terminal arteriole was 100% of baseline in the RIPC group compare to 83% in Sham RIPC [P values < 0.05]. Capillary reperfusion was 83% of baseline in the cremaster pretreated with RIPC compared to 60% in sham RIPC [P value < 0.05]. Norepinephrine (10-6 M) induced vasoconstriction was completely reversed by acetylcholine (10-7M) in the cremaster pretreated with RIPC compared to a very limited vasodilatation occurring in sham RIPC [P value < 0.05]. Systemic intravenous infusion of adenosine produced profound hypotension and was unable to mimic RIPC effect against prolonged ischemia in the cremaster muscle. Local intra-arterial infusion of SPT into cremaster muscle was also unable to block microvascular protection induced by RIPC from left hindlimb. Conclusion: The microvascular protection induced by remote ischemic preconditioning is not mediated by adenosine.