Interstitial cells of Cajal (ICC) and the enteric nervous system orchestrate the various rhythmic motor patterns of the colon. Excitation of ICC may evoke stimulus-dependent pacemaker activity and will therefore have a profound effect on colonic motility. The objective of the present study was to evaluate the potential role of K(+) channels in the regulation of ICC excitability. We employed the cell-attached patch clamp technique to assess single channel activity from mouse colon ICC, immunohistochemistry to determine ICC K(+) channel expression and single cell RT-PCR to identify K(+) channel RNA. Single channel activity revealed voltage-sensitive K(+) channels, which were blocked by the KV7 blocker XE991 (n = 8), which also evoked inward maxi channel activity. Muscarinic acetylcholine receptor stimulation with carbachol inhibited K(+) channel activity (n = 8). The single channel conductance was 3.4 ± 0.1 pS (n = 8), but with high extracellular K(+), it was 18.1 ± 0.6 pS (n = 22). Single cell RT-PCR revealed Ano1-positive ICC that were positive for KV7.5. Double immunohistochemical staining of colons for c-Kit and KV7.5 in situ revealed that intramuscular ICC (ICC-IM), but not ICC associated with the myenteric plexus (ICC-MP), were positive for KV7.5. It also revealed dense cholinergic innervation of ICC-IM. ICC-IM and ICC-MP networks were found to be connected. We propose that the pacemaker network in the colon consists of both ICC-MP and ICC-IM and that one way of exciting this network is via cholinergic KV7.5 channel inhibition in ICC-IM.