Interstitial cells of Cajal (ICC) are pacemaker cells for gut peristaltic motor activity. Little is known about mechanisms that regulate ICC excitability. The objective of the present study was to investigate a potential role for clotrimazole- (CTL-) sensitive K currents in the regulation of ICC excitability and pacemaker activity. ICC were studied in situ and in short term culture using the whole-cell patch clamp configuration. In situ, ICC exhibited spontaneous transient inward currents followed by transient outward currents. CTL blocked outward currents thereby increasing the net inward currents and depolarized ICC thereby establishing CTL-sensitive channels as regulators of ICC pacemaker activity. In short term culture, a clotrimazole-sensitive K⁺ current (I_CTL) was identified that showed increased conductance when depolarized from the resting membrane potential to 0 mV and subsequent inward rectification at further depolarized potentials. The I_CTL markedly increased with increasing intracellular calcium and was insensitive to the ERG blocker E4031 and the BK_Ca blocker iberiotoxin. I_CTL contributed 3 - 9 nS to the whole cell conductance at 0 mV membrane potential under physiological conditions, it was fast activating ( = 88 ms), showed little time dependent inactivation and exhibited a de-activation time constant of 38 ms. The nitric oxide donor, sodium nitroprusside (SNP), increased I_CTL. Single channel activity, activated by calcium and SNP, was inhibited by CTL, with a single channel conductance of ~ 38 pS. In summary, ICC generate an I_CTL upon depolarization through an intermediate conductance K_Ca channel that regulates pacemaker activity and ICC excitability.