The role of adenosine and contraction for secretion of vascular endothelial growth factor (VEGF) in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted in the thigh muscle of seven male subjects, and dialysate was collected at rest, during infusion of adenosine, and during knee extensor exercise. The dialysate was analyzed for content of VEGF protein and adenosine. The mechanism of VEGF secretion from muscle cells in culture was examined in resting and electrostimulated cells and in response to the adenosine analog NECA and the adenosine A(2A) receptor specific analog CGS-21680. Adenosine receptors A(1), A(2A), and A(2B) were blocked with DPCPX, ZM-241385, and enprofylline, respectively. cAMP-dependent protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) were inhibited by H-89 and PD-98509, respectively. The human experiment showed that adenosine infusion enhanced (P < 0.05) the interstitial concentration of VEGF protein approximately fourfold above baseline. Exercise increased (P < 0.05) the interstitial VEGF concentration approximately sixfold above rest in parallel with an approximately threefold increase in adenosine concentration. In accordance, in cultured muscle cells, NECA and contraction caused secretion of VEGF (P < 0.05). The contraction-induced secretion of VEGF was abolished by the A(2B) antagonist enprofylline and by inhibition of PKA or MAPK. The results demonstrate that adenosine causes secretion of VEGF from human skeletal muscle cells and that the contraction-induced secretion of VEGF protein is partially mediated via adenosine acting on A(2B) adenosine receptors. Moreover, the contraction-induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK activation, but only the MAPK pathway appears to be adenosine dependent, revealing involvement of additional pathways in VEGF secretion.