The erythrocyte is proposed to play a key role in the control of local tissue perfusion via three O 2-dependent signaling mechanisms: 1) reduction of circulating nitrite to vasoactive NO, 2) S-nitrosohemoglobin (SNO-Hb)-dependent vasodilatation, and 3) release of the vasodilator and sympatholytic ATP; however, their relative roles in vivo remain unclear. Here we evaluated each mechanism to gain insight into their roles in the regulation of human skeletal muscle blood flow during hypoxia and hyperoxia at rest and during exercise. Arterial and femoral venous hemoglobin O 2 saturation (O 2Hb), plasma and erythrocyte NO and ATP metabolites, and leg and systemic hemodynamics were measured in 10 healthy males exposed to graded hypoxia, normoxia, and graded hyperoxia both at rest and during submaximal one-legged knee-extensor exercise. At rest, leg blood flow and NO and ATP metabolites in plasma and erythrocytes remained unchanged despite large alterations in O 2Hb. During exercise, however, leg and systemic perfusion and vascular conductance increased in direct proportion to decreases in arterial and venous O 2Hb (r 2 = 0.86-0.98; P = 0.01), decreases in venous plasma nitrite (r 2 = 0.93; P < 0.01), increases in venous erythrocyte nitroso species (r 2 = 0.74; P < 0.05), and to a lesser extent increases in erythrocyte SNO (r 2 = 0.59; P = 0.07). No relationship was observed with plasma ATP (r 2 = 0.01; P = 0.99) or its degradation compounds. These in vivo data indicate that, during low-intensity exercise and hypoxic stress, but not hypoxic stress alone, plasma nitrite consumption and formation of erythrocyte nitroso species are associated with limb vasodilatation and increased blood flow in the human skeletal muscle vasculature. Copyright © 2010 the American Physiological Society.