During exercise, blood flow to active skeletal muscle is increased to precisely meet the oxygen demand required for energy production. The precise regulation of blood flow is overall achieved through a balance between constriction via sympathetic nervous activity, locally formed vasoconstrictors and vasodilators and compounds that can modulate the constrictive effect of the sympathetic nervous system (functional sympatholysis). The regulation of local formation of vasoactive and sympatholytic compounds in the muscle is complex and not yet fully understood but a number of mechanisms and cells have been proposed to contribute. Central for most vasodilator mechanisms are the endothelial cells that receive signals and respond to chemical and mechanical stimuli by hyperpolarization and release of vasodilators including nitric oxide (NO) and prostacyclin. The chemically induced stimulation is achieved by compounds such as ATP and adenosine, originating from erythrocytes in response to oxygen desaturation of the hemoglobin molecule and, on the interstitial side, from skeletal muscle cells in response to contraction. Interestingly ATP is also a potent sympatholytic compound. A strong mechanical stimulus particularly for NO formation, is the frictional force that the blood exerts on the endothelial membrane (shear stress) which is sensed and transduced by mechanosensors. Substantial advancements in the area of skeletal muscle blood flow regulation have been made in the recent years but key aspects that remain to be resolved are the mechanisms underlying the close coupling between oxygen demand and regulation of blood flow and fully understanding functional sympatholysis.