Materials and Methods: Previously, we have reported that the adenosine(ADO)-initiated microvascular protection in the late phase of ischemic preconditioning (IPC) can be blocked by an inhibitor of nitric oxide synthase. The purpose of present study was to determine eNOS and iNOS gene expression and microvascular response in the cremaster muscle 24 hours after ischemic- or ADO-preconditioning. Vascular isolated cremaster model in male SD rats was used. IPC consisted of 45 min of ischemia on day 1 and then 24 hours of reperfusion. To minic the effects of IPC in the late phase, ADO (0.35mg/100g) was given via local intra-arterial infusion at a rate of 0.1ml/h for 15 mins. To block the effects of IPC in the late phase, 8-sulfophenyl-theophylline (SPT; a nonspecific ADO receptor blocker; 0.625mg/100g) was given in the same way prior to IPC. Two parts of study have been done. In the microcirculation aspect, cremaster muscles underwent 4-hours of warm ischemia and then 60-minutes of reperfusion on day 2. Microvascular responses in the cremaster muscle to IPC or pharmacological preconditioning were determined by measuring terminal arteriole diameter and capillary perfusion using intravital microscopy and by the evaluation of the endothelium-dependent and -independent vasodilatations in terminal arterioles. In the molecular biology aspect, cremaster muscles were harvested on day 2 for measuring eNOS and iNOS gene expression by semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (QRT-PCR). Four groups (12 rats/per group) have designed. Group1: Sham IPC, Group 2: IPC, Group3: ADO+Sham IPC, Group4: SPT+IPC. Results: The average diameter of terminal arterioles after 4 hrs ischemia/reperfusion was significantly larger in IPC (121% of baseline) or ADO group (113%) than sham IPC (71%) or SPT group (79%)(P<0.05). The average capillary perfusion was also better in IPC group (96% of baseline) or ADO group (105%) than Sham IPC group (54%) or in SPT group (65%)(P<0.05). Endothelium-dependent vasodilatation induced by acetylcholine (10-7M) was shown intact in IPC or ADO pre-treated cremasters, but impaired in sham IPC or SPT pre-treated cremasters. IPC or ADO preconditioning on day 1 significantly enhanced eNOS mRNA expression (30% or 15% increase from control respectively) and iNOS mRNA expression (15 or 20% increase from control respectively) on day 2(P<0.05). Conclusion: The results from these studies suggest that ischemic or ADO preconditioning induces late phase microvascular protection in the skeletal muscle by a NOSs-dependent mechanism.