We sought to delineate the effects of acute and chronic exercise on the regulation of intracellular nitric oxide (NO(i)) production in putative endothelial progenitor cells (EPCs). Putative EPC colony-forming units (CFU-EC) were cultured from blood drawn before and after 30 min of treadmill exercise at 75% of maximal oxygen uptake in active (n = 8) and inactive (n = 8) men. CFU-EC were similar between groups at baseline, but increased after exercise in active men only (P = 0.04). CFU-EC expressed lower NADPH oxidase subunit gp91(phox) mRNA and elevated endothelial nitric oxide synthase mRNA in active relative to inactive men at baseline (P < 0.05). Acute exercise reduced gp91(phox) mRNA in CFU-EC of both groups (P < 0.05), whereas p47(phox) mRNA levels were reduced in the inactive group only (P = 0.02). There were no differences between groups or with acute exercise in xanthine oxidase, superoxide dismutase isoforms, or gluthathione peroxidase-1 mRNA levels. NO(i) was significantly greater in CFU-EC of active men at baseline (P = 0.004). NO(i) increased in CFU-EC of inactive men with acute exercise, and in vitro experiments with apocynin indicated the increased NO(i) production was caused by suppression of NADPH oxidase. However, the increases in NO(i) with the different treatments in the inactive group did not reach the baseline levels in the active group (P < 0.05). We conclude that acute exercise increases NO(i) in cells generated by the CFU-EC assay through an NADPH oxidase-inhibition mechanism in sedentary men. However, differences due to chronic exercise must involve additional factors. Our findings support exercise as a means to improve putative EPC function and suggest a novel mechanism that may explain this effect.