The role of adenosine and contraction for secretion of VEGF in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted into 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 electro stimulated cells, and in response to the adenosine analogue NECA, and the adenosine A(2A) receptor specific analog CGS21680. Adenosine receptors A(1), A(2A) and A(2B) were blocked with DPCPX, ZM241385 and enprofyllin, 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 ~ 4-fold above baseline. In comparison, exercise increased (P<0.05) the interstitial VEGF concentration ~6-fold above rest in parallel with a ~3-fold 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 protein was abolished by the A(2B) antagonist enprofyllin and markedly reduced 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 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.