To investigate mechanisms underlying the transmission of spontaneous Ca signals in the bladder, changes in intracellular concentrations of Ca²⁺ ([Ca²⁺]_i) were visualized in isolated detrusor smooth muscle bundles of the guinea-pig urinary bladder loaded with a fluorescent Ca²⁺ indicator, fura-PE3 or fluo-4. Spontaneous increases in [Ca²⁺]_i (Ca transients) preferentially originated along the boundary of muscle bundles and then spread to the other boundary (Ca waves). The synchronicity of Ca waves across the bundles was disrupted by either 18-glycyrrhetinic acid (18-GA, 40 µM), carbenoxolone (30 µM) or 2-aminoethoxydiphenylborate (2-APB, 50 - 100 µM), while CPA (10 µM), ryandine (100 µM), xestospongin C (3 µM) and U-73122 (10 µM) had no effect. Intracellular recordings using two independent microelectrodes demonstrated that 2-APB (100 µM) blocked electrical coupling between detrusor smooth muscle cells. Nifedipine (10 µM) but not nominal Ca²⁺ free solution diminished the synchronicity of Ca waves before preventing their generation. Staining for c-kit identified interstitial cells (IC) located along both boundaries of muscle bundles. IC were also scattered amongst smooth muscle cells and distributed more dominantly in connective tissue between muscle bundles. IC generated nifedipine-resistant spontaneous Ca transients which occurred independently from those of smooth muscles. In conclusion, the propagation of Ca signals appears to be exclusively mediated by the spread of action potentials through gap junctions being facilitated by the regenerative nature of L-type Ca channels without significant contribution of intracellular Ca stores. IC in the bladder may modulate the transmission of Ca transients originating from smooth muscle cells rather than be the pacemaker of spontaneous activity.