Vascular ATP-sensitive potassium (K_ATP) channels have an important role in hypoxic vasodilation. Since K_ATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured K_ATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from rat femoral artery using the whole cell patch clamp technique. K_ATP current was not activated by exposure of cells to hypoxic solutions (pO₂~35 mmHg). In contrast, voltage-dependent calcium current and the depolarisation-induced rise in intracellular calcium concentration ([Ca²⁺]_i) was inhibited by hypoxia. Blocking mitochondrial ATP production using the ATP synthase inhibitor oligomycin B (3 µM) did not activate current. Blocking glycolytic ATP production using 2-deoxy-D-glucose (2-DG, 5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP, 1 µM) depolarised the mitochondrial membrane potential and activated K_ATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarised the mitochondrial membrane potential and activated K_ATP current. Our data show that: 1. Anoxia but not hypoxia activates K_ATP current in femoral artery myoctes: 2. Inhibition of cellular energy production is insufficient to activate K_ATP current, and that energy consumption is required for current activation. These results suggest vascular K_ATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca²⁺]_i through modulation of calcium channel activity.