Urethral smooth muscle cells (USMC) sustain tonic contractions to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral tissues and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit⁺ interstitial cells of Cajal (ICC), gastrointestinal pacemakers and neuroeffectors. Isolated cell studies of urethral ICC-like cells (ICC-LC) exhibit spontaneous intracellular Ca²⁺ signalling behaviours linked to proposed roles as USMC pacemakers and neuromodulators similar to ICC in the gut, although observation and direct stimulation of ICC-LC within intact urethral tissues is lacking.  Using a mouse line with cell-specific expression of the Ca²⁺ indicator GCaMP6f, driven off the Kit promoter (Kit-GCaMP6f mice), we unequivocally identified ICC-LC within in situ urethra preparations and characterized their activity. Across 54 cells imaged from 13 animals, ICC-LC fired spontaneous Ca²⁺ waves that propagated on average 40.1 ± 0.7 mm, with varying amplitudes (0.08 – 1.96 DF/F₀), which originated from multiple firing sites per cell (average 3.1 ± 0.2). ICC-LC imaged from Kit-GCaMP6f urethra did not form interconnected networks. ICC-LC activity was uncoordinated across multiple cells with no obvious entrainment of ICC-LC. ICC-LC Ca²⁺ event frequency was unaffected by the L-type Ca²⁺ channel inhibitor nifedipine (P=0.25, n=5) but was abolished by cyclopiazonic acid (P=0.0005, n=6) and decreased by an inhibitor of store-operated Orai Ca²⁺ channels (GSK-7975A, P<0.0001, n=6). While the a-adrenoceptor agonist phenylephrine increased Ca²⁺ wave frequency (P=0.0007), amplitude (P=0.04) and spread (P=0.14) (n=6), the nitric oxide (NO) donor DEA-NONate had no effect (n=7). Electrical field stimulation (EFS, 10 Hz) of intrinsic nerves under excitatory (n=6) or inhibitory conditions (n=7) failed to elicit responses in ICC-LC. In contrast, EFS of ICC from Kit-GCaMP6f colon yielded consistent excitatory (cholinergic, n=3) and inhibitory (nitrergic, n=5) postjunctional responses. We conclude urethral ICC-LC are spontaneously active but are not functionally innervated.