The aims of the present study were to characterise the location and morphology of ICC in the urethra and bladder and to examine their structural relationships with nerves and smooth muscle. Also, to investigate the physiological role of ICC in the normal bladder by investigating the Ca2+ signalling in smooth muscle cells and ICC, in in situ mucosal preparations of guinea pig bladder, using ca2+~imaging techniques. Rabbit urethras were fixed, labelled with antibodies and examined with confocal microscopy. Staining with Masson's Trichrome showed the arrangement of smooth muscle bundles into distinct layers. Anti-c-kit revealed a population of immuno-positive ICC, which were either elongated with several lateral branches or stellate with branches coming from a central soma. ICC were found throughout the muscularis. c-kit-positive ICC were found on the boundary of the myosin-positive smooth muscle bundles, and also in the spaces between. Anti-c-kit and anti-neurofilament showed frequent points of close contact between the c-kit-positive ICC and nerves some of which were immunopositive for nNos. Spontaneous Ca2+ transients were investigated within the live guinea pig bladder using Oregon green Ca2+ indicator. Spontaneous smooth muscle Ca2+ transients took two forms, uncoordintated single cell Ca2+ events, and coordinated whole bundle flashes. ICC generated Ca2+ transients had longer durations and were significantly less frequent than smooth muscle cell events. There was little evidence of correlation between the spontaneous Ca2+ transients of smooth muscle cells and those of ICC. Both the smooth muscle cell and ICC activity were affected by various store and channel blockers. In conclusion, ICC were located throughout the wall of the rabbit urethra, where they came into close contact with smooth muscle cells and nerves. ICC could have a role in the excitation of smooth muscle cells in the urinary bladder, providing a possible therapeutic target for many urinary tract pathologies.