To investigate the possible involvement of outward potassium (K+) currents in nitric oxide-induced relaxation in intestinal smooth muscle, we used whole-cell patch clamp technique in freshly dispersed guinea-pig ileum longitudinal smooth muscle cells. When cells were held at ⁣60 mV and depolarized from ⁣40 mV to ⁢50 mV in 10 mV increments, sustained outward K+ currents were evoked. The outward K+ currents were markedly increased by the addition of 10 ㄍM sodium nitroprusside (SNP). 10 ㄍM S-nitroso-N-acetylpenicillamine (SNAP) and 1 mM 8-Bromo-cyclic GMP (8-Br-cGMP) also showed a similar effect to that of SNP. 1 mM tetraethylammonium (TEA) significantly reduced depolarization-activated outward K+ currents. SNP-enhanced outward K+ currents were blocked by the application of TEA. High EGTA containing pipette solution (10 mM) reduced the control currents and also inhibited the SNP-enhanced outward K+ currents. 5 mM 4-aminopyridine (4-AP) significantly reduced the control currents but showed no effect on SNP-enhanced outward K+ currents. 0.3 ㄍM apamin and 10 ㄍM glibenclamide showed no effect on SNP-enhanced outward K+ currents. 10 ㄍM 1H-[1,2,4]oxadiazolo [4,3-a]quinoxaline-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase, significantly blocked SNP-enhanced K+ currents. We conclude that NO donors activate the Ca2+-activated K+ channels in guinea-pig ileal smooth muscle via activation of guanylate cyclase.