In the present study we examined whether exercise and prostanoids have an effect on the muscle interstitial concentration of vascular endothelial growth factor (VEGF) and on the proliferative effect of muscle interstitial fluid. Dialysate from resting and exercising human skeletal muscle, obtained either during control conditions or during cyclooxygenase inhibition, was examined for its content of VEGF and for its effect on endothelial cell proliferation. Microdialysis probes with high (960 kDa) and low (5 kDa) molecular-mass cut-off membranes were placed in the vastus lateralis muscle of healthy young males. The subjects performed one-legged knee extensions (20 W). The concentration of VEGF in the 960 kDa dialysate was greater (P < 0.05) during exercise compared to at rest (67 ± 28 vs. 230 ± 22 pg ml−1). The rate of endothelial cell proliferation was 2.7-fold higher (P < 0.05) with the 960 kDa dialysate from resting muscle than with perfusate and was 5.8-fold higher (P < 0.05) than the perfusate value with dialysate from exercising muscle. VEGF was not enhanced with exercise in the 5 kDa dialysate, yet the exercise dialysate induced a 1.9-fold higher (P < 0.05) proliferation than the resting dialysate. Cyclooxygenase inhibition did not affect the VEGF concentration or the proliferating effect of the dialysates (P > 0.05). This study demonstrates for the first time that VEGF is present in the interstitium of human skeletal muscle and that exercise enhances the interstitial concentration of VEGF and of other, as yet unidentified, angiogenic compounds. Products of cyclooxygenase do not appear to have an effect on the release of VEGF or other proliferative agents in human skeletal muscle.