Chronic hypoxia (CH) results in an endothelium-dependent blunting of myogenic responsiveness in isolated rat skeletal muscle arteries. We hypothesized that this attenuated myogenic responsiveness is due to an alteration of large conductance Ca2+-activated K+ (BKCa) channel activity in the endothelial cell of CH arteries. Third order branches of the caudal femoral artery (150–200 μm) from normoxic control rats and rats exposed to hypoxia (PB = 380 mmHg for 48 hours), were isolated, cannulated and pressurized. Vessel inner diameter was recorded at intraluminal pressures of 20–200 mmHg under normoxic conditions. Blunted myogenic responsiveness of CH arteries persisted in the presence of N-Nitro-L-arginine (L-NNA) (100 μM) and indomethacin (10 μM) and after further treatment with the luminally administered small and intermediate conductance potassium channel blockers, apamin (100 nM) and TRAM 34 (1 μM), respectively. However, myogenic responsiveness of CH arteries was restored to control levels by luminal administration of either a combination of charybdotoxin (100 nM) and apamin (100 nM) (n=5, both groups) or iberiotoxin (100 nM) alone (n=5, both groups), in the continued presence of L-NNA and indomethacin. These latter treatments did not affect myogenic reactivity in arteries from control rats. We conclude that endothelial cell BKCa channel activity is increased in CH arteries resulting in hyperpolarization that is conducted to the underlying vascular smooth muscle thereby attenuating myogenic responsiveness.