Background: Large-conductance calcium-activated potassium channels (BKCa) of vascular smooth muscle cells (VSMCs) play an important role in the regulation of vascular tone. We tested the hypotheses that the alteration of arterial relaxation after exposure to ischemia or hyperkalemia is attributable to the alteration in the activities of BKCa by using the patch-clamp technique and force measurement. Methods: VSMCs were enzymatically isolated from porcine coronary arteries, with the primary cultures used for patch-clamp study. BKCa currents were recorded in a whole-cell configuration (n = 5 in each group). The BKCa activator NS1619 (-7 to -4.5 logM)-induced relaxation was studied myographically in small porcine coronary arteries (n = 6 per group). The effects of global ischemia for 1 hour and hyperkalemia (20 to 120 mmol/liter) were tested. Results: Ischemia for 1 hour markedly reduced the BKCa current from 119.8 ± 11.4 pA/pF to 86.0 ± 3.7 pA/pF (p < 0.05). The rise of extracellular K+ resulted in increased BKCa currents in a concentration-dependent manner (5.4 mmol/liter: 31.6 ± 3.4 pA/pF; 20 mmol/liter: 73.6 ± 11.4 pA/pF; 60 mmol/liter: 108.6 ± 20.6 pA/pF; 120 mmol/liter: 135.2 ± 20.5 pA/pF; p < 0.05). NS1619-induced relaxation was suppressed by ischemia (71.4 ± 2.2% vs 95.3 ± 1.6%; p < 0.01) and was inferior in K+ pre-contraction, as compared with U46619 (a thromboxane A2 mimetic) pre-contraction (p < 0.05). Conclusions: In coronary circulation: (1) the BKCa current density and related vasorelaxation are reduced after ischemia; and (2) hyperkalemia induces dual effects; the depolarization obscures the compensatory increase of the BKCa activity. These findings imply that BKCa activators may be used in cardioplegia or heart preservation solutions to protect the function of this ion channel. © 2009 International Society for Heart and Lung Transplantation.