Studies of enzymatically isolated cells from the rabbit urethra have established that there is a population of specialised cells which display spontaneous electrical activity and have morphological properties of interstitial cells (Sergeant et al. 2000). The aim of the present study was to identify such cells within the wall of the rabbit urethra using antibodies to the Kit receptor and examine their location, morphology and relationships with nerves and smooth muscle. Urethras were obtained post mortem from rabbits. Tissues were labelled with antibodies and examined with confocal microscopy. Some specimens were embedded in paraffin and processed for histological staining. Histological sections from the most proximal third and mid-third region of rabbit urethra were stained with Casson’s Trichrome to reveal their cellular arrangement. Sections from both regions had outer longitudinal and inner circular layers of smooth muscle and a lamina propria containing connective tissue and blood vessels. The lumen was lined with urothelial cells. The mid-third region had a better developed circular smooth muscle layer compared to the most-proximal samples and had extensive inner longitudinal smooth muscle bundles in the lamina propria. Labelling with anti-c-Kit revealed a population of Kit-positive cells within the wall of the rabbit urethra (n=15). These were found in the circular and longitudinal layers of the muscularis. Double-labelling with anti-smooth muscle myosin (n=13) showed Kit-positive cells on the boundary of the smooth muscle bundles, orientated in parallel with the axis of the bundles. Others were found in the spaces between the bundles and often made contact with each other, forming small network areas. The Kit-positive cells had a number of morphologies: elongated with several lateral branches or stellate with branches coming from a central nuclear region. Relationships with intramural nerves was examined by co-labelling with anti-neurofilament (n=5). Frequent points of contact were observed between the Kit-positive cells and nerves. Similar observations were made with co-labelling with anti-neuronal nitric oxide synthase (n=5). In conclusion, Kit-positive interstitial cells are found within the smooth muscle layers of the rabbit urethra. These are located on the edge of smooth muscle bundles and in the spaces between the bundles and make structural associations with nerves. The contact with nNOS containing neurons might imply participation of the interstitial cells in the nitrergic inhibitory neurotransmission of the urethra.