Cyclic nucleotide-gated (CNG) channels are non-selective cation channels that mediate influx of extracellular Na+ and Ca2+ in various cell types. L-cis-Diltiazem, a CNG channel blocker, inhibits contraction of urethral smooth muscle (USM), however the mechanisms underlying this effect are still unclear. We investigated the possibility that CNG channels contribute to spontaneous pacemaker activity in freshly isolated interstitial cells of Cajal (ICC) isolated from the rabbit urethra (RUICC). Using immunocytochemistry, we found intense CNG1-immunoreactivity in vimentin-immunoreactive RUICC, mainly within patches of the cellular body and processes. In contrast, α-actin immunoreactive smooth muscle cells (SMC) did not show significant reactivity to a specific CNGA1 antibody. Freshly isolated RUICC, voltage clamped at −60 mV, developed spontaneous transient inward currents (STICs) that were inhibited by L-cis-Diltiazem (50 μM). Similarly, L-cis-Diltiazem (50 μM) also inhibited Ca2+ waves in isolated RUICC, recorded using a Nipkow spinning disk confocal microscope. L-cis-Diltiazem (50 μM) did not affect caffeine (10 mM)-induced Ca2+ transients, but significantly reduced phenylephrine–evoked Ca2+ oscillations and inward currents in in RUICC. L-type Ca2+ current amplitude in isolated SMC was reduced by ˜18% in the presence of L-cis-Diltiazem (50 μM), however D-cis-Diltiazem, a recognised L-type Ca2+ channel blocker, abolished L-type Ca2+ current but did not affect Ca2+ waves or STICs in RUICC. These results indicate that the effects of L-cis-diltiazem on rabbit USM could be mediated by inhibition of CNG1 channels that are present in urethral ICC and therefore CNG channels contribute to spontaneous activity in these cells.