We examined whether the formation or the release of the vasodilators adenosine, prostacyclin (PGI<sub>2</sub>) and potassium (K<sup>+</sup>) increase in skeletal muscle interstitium in response to nitric oxide synthase (NOS) inhibition. Five subjects performed one-legged knee extensor exercise at 30 W without (controls) and with prior N<sup>G</sup>-nitro- L-arginine methyl ester (L-NAME) infusion (4 mg/kg, intravenously). Samples from the interstitial fluid were obtained at rest, during exercise and after exercise with the microdialysis technique. Interstitial adenosine in controls increased (p < 0.05) from 0.11 ± 0.03 μmol/l at rest to 0.48 ± 0.06 μmol/l during exercise. Interstitial adenosine during exercise in L-NAME was similar (p > 0.05) to controls. The 6-keto-prostaglandin F1α concentration in controls was 1.17 ± 0.20 ng/ml at rest and increased (p < 0.05) to 1.97 ± 0.30 ng/ml during exercise and was further elevated (p < 0.05) to 2.76 ± 0.38 ng/ml after exercise and these concentrations were not different (p > 0.05) in L-NAME. The interstitial K<sup>+</sup> concentration in controls increased (p < 0.05) from 4.1 ± 0.1 mmol/l at rest to 9.5 ± 0.5 mmol/l during exercise. The interstitial K<sup>+</sup> concentration during exercise (6.7 ± 0.4 mmol/l) was lower (p < 0.05) in L-NAME than in controls. The present findings demonstrate that the muscle interstitial concentrations of adenosine, PGI<sub>2</sub> and K<sup>+</sup> during exercise are not increased with systemic NOS inhibition. Thus, the lack of effect of NOS inhibition on the rate of blood flow to contracting human skeletal muscle does not appear to be due to compensatory formation or release of adenosine, PGI<sub>2</sub> and K<sup>+</sup> in the muscle interstitium. The present study also supports a role for PGI<sub>2</sub> in the regulation of blood flow during exercise.