Interstitial cells of Cajal (ICC) are pacemaker cells for gut peristaltic motor activity. Compared with cardiac pacemaker cells, 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 ( ICTL) in the regulation of ICC excitability and pacemaker activity. ICC were studied in situ and in short-term culture by 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 ICTL 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 ICTL markedly increased with increasing intracellular calcium and was insensitive to the ether-à-go-go-related K channel blocker E-4031 and the large-conductance calcium-activated K channel blocker iberiotoxin. ICTL contributed 3–9 nS to the whole cell conductance at 0 mV membrane potential under physiological conditions; it was fast activating (τ = 88 ms), showed little time-dependent inactivation, and exhibited a deactivation time constant of 38 ms. The nitric oxide donor sodium nitroprusside (SNP) increased ICTL. Single-channel activity, activated by calcium and SNP, was inhibited by CTL, with a single-channel conductance of ∼38 pS. In summary, ICC generate a ICTL on depolarization through an intermediate-conductance calcium-activated K channel that regulates pacemaker activity and ICC excitability.