Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulus, the expression of which increases in skeletal muscle after exercise. Because exercise is also accompanied by increased intramuscular reactive oxygen species (ROS) generation, we tested the hypothesis that ROS stimulate VEGF production from skeletal myotubes. Differentiated C(2)C(12) skeletal myotubes exposed to ROS-producing agents exhibited a concentration-dependent increase in VEGF production, whereas undifferentiated myoblasts did not respond to oxidants. Moreover, conditioned medium from ROS-treated myotubes increased the bovine lung microvascular cell proliferation rate. To study the mechanism(s) involved in the stimulation of VEGF production by ROS, myotubes were pretreated with a selective phosphatidylinositol 3-kinase (PI3K) inhibitor, LY-294002, before being exposed to hydrogen peroxide or pyrogallol. LY-294002 attenuated both Akt phosphorylation and VEGF production. In addition, oxidants increased nuclear factor-kappaB-dependent promoter activity in transiently transfected myotubes; however, pretreatment with the pharmacological inhibitor of nuclear factor-kappaB, diethyldithiocarbamate, did not affect the oxidant-stimulated VEGF release. We conclude that ROS induce VEGF release from myotubes via a PI3K/Akt-dependent pathway.