Prolonged ischemia and oxygen-derived free radical causing ischemic-reperfusion injury are believed to be among the major factors responsible for microsurgical flap failure. Increasing flap tolerance to ischemic-reperfusion injury may enhance the ability to salvage compromised flaps. Ischemic preconditioing has been found to be effective in decreasing muscle functional impairment after prolonged ischemia. NO is a critical factor related with free radical production in ischemic-reperfusion injury. In this study we examined the molecular marker of NO to determine its role in correlation with ischemic preconditioning in the muscle flap survival in a rat model. Fifty male Sprague-Dawley rats were used in the study. The gracilis muscle flap with femoral vascular pedicle was used as the flap model. The ischemic preconditioning consisted of three sequences of clamping the pedicle for 10 minutes followed by 10 minutes of reperfusion for a total of 1 hour. In part I, the experimental group (n=10) underwent ischemic preconditioning for 1 hour. In the control group (n=10), the flaps were dissected without clamping of the pedicle. Both groups were then subjected to 4 hours of global ischemia by continuous pedicle clamping, after which the flaps were sutured to their beds. On postoperative day 3, survival of the flaps was determined by gross and histologic examinations. In part II, the experimental group (n=12) underwent ischemic preconditioning while the control group (n=12) did not. The flaps from each group were harvested for iNOS gene expression using RT-PCR at the end of 1 hour after reperfusion and 4 hours of global ischemia. The results indicated a significantly higher survival rate in the experimental group than in the control group (90 vs. 50 percent, p<0.05). iNOS gene expression was significantly higher in the experimental group than the control group at 1 hour after ischemic preconditioning (0.73± 0.18 vs. 0.26± 0.11, p<0.01). However, after 4 hours of global ischemia, iNOS expression in the control group was statistically higher than the experimental group (0.83± 0.16 vs. 0.26± 0.07, p<0.01). We conclude that ischemic preconditioning can enhance flap tolerance to ischemia-reperfusion injury and improve flap viability rate. This study provides evidence that the regulation of NOS may play a role in ischemic preconditioning phenomenon and warrants further investigation.