The human hypoxia inducible factor 1 (HIF-1) system is activated under various pathological conditions, yet less is known about its physiological regulation in healthy human tissue. We have studied the effect of exercise on the activation of HIF-1 in human skeletal muscle. Employing a model where oxygen consumption increases and oxygen tension can be manipulated, nine healthy male subjects performed 45 min of one-legged knee-extension exercise. Biopsies were taken before, directly after, and 30, 120, and 360 min after exercise. Exercise led to elevated HIF-1alpha protein levels and a more prevalent nuclear staining of HIF-1alpha. Interestingly, a concurrent decrease in von Hippel-Lindau tumor suppressor protein (VHL) levels was detected in some subjects. Moreover, exercise induced an increase in the DNA binding activity of HIF-1alpha. Characterization of gene expression by real-time PCR demonstrated that the HIF-1 target genes VEGF and EPO were activated. VEGF mRNA was further increased when blood flow to the exercising leg was restricted. In conclusion, these data clearly demonstrate that physical activity induces the HIF-1-mediated signaling pathway in human skeletal muscle, providing the first evidence that human HIF-1alpha can be activated during physiologically relevant conditions.