The aim of this study was to clarify the mechanism of the inhibitory action of carbon monoxide (CO) on contraction, by measuring cytosolic Ca2⁢ level ([Ca2⁢]i) and ionic currents in guinea-pig ileum. CO (10%) inhibited 40 mM KCl-induced contraction and this effect was blocked by ODQ (1μM), a soluble guanylyl cyclase (sGC) inhibitor. CO inhibited the 40 mM KCl-induced contraction without changing [Ca2⁢]i. Cumulative addition of KCl induced a graded increase in [Ca2⁢]i and muscle tension. In the presence of CO, cumulative addition of KCl induced smaller contraction than in the absence of CO. On the other hand, the increase in [Ca2⁢]i induced by cumulative addition of KCl was only slightly decreased in the presence of CO, and the [Ca2⁢]i-tension relationship shifted downwards. Using the patch clamp technique with a holding potential of ⁣60 mV, we found that CO had little effect on the peak Ba currents (IBa) when voltage was stepped from ⁣60 mV to 0 mV. In addition, CO showed no effect on the depolarization-activated outward K⁢ currents in the all potential ranges. We conclude that CO inhibits smooth muscle contraction mainly by decreasing the Ca2⁢ sensitivity of contractile elements via a cGMP-dependent pathway, not by involving L-type Ca2⁢ and outward-potassium currents in guinea-pig ileum.