The cellular mechanisms underlying pathological bladder conditions including urge urinary incontinence are poorly understood. An increasing body of evidence suggests that interstitial cells of Cajal (ICC) may have significant roles in normal bladder activity (Brading & McCloskey, 2005) and also in conditions leading to overactivity (Kubota et al, 2008). Bladder ICC have morphological similarities to ICC of the gastrointestinal tract and have been identified with the established marker, anti-c-Kit, in animal tissues (Davidson & McCloskey, 2005). The purpose of the present study was to identify ICC in human bladder samples and examine their relationships with nerves and smooth muscle. Bladder biopsies were obtained with informed consent and full ethical approval from patients undergoing urological investigation. Whole-mount, flat sheet tissue preparations were labeled with antibodies using standard immunohistochemical protocols and imaged with a confocal microscope. Single cells were dissociated from biopsy samples using a cocktail of enzymes and viewed with bright-field microscopy. Tissues labeled with anti-c-Kit had several sub-populations of immunopositive cells (n=18 samples). c-Kit-positive cells in the lamina propria were branched, stellate-shaped with a central nucleus and formed frequent connections with each other. When the biopsy samples contained underlying detrusor smooth muscle bundles, c-Kit-positive cells with lateral branches from an elongated cell body were located on the boundary of the bundles. In some biopsies, discrete, rounded, non-branched c-Kit-positive cells were present; these were deemed to be mast cells. Double-labeling with anti-c-Kit and anti-vAChT to label cholinergic nerves demonstrated close relationships between ICC and cholinergic nerves (n=4 samples) in both the lamina propria and the underlying detrusor. Vimentin-positive cells were abundant throughout the lamina propria and formed a network of interconnecting cells with different morphologies including stellate and elongated bipolar cells (n=4 samples). Vimentin filaments are present in fibroblasts, ICC and other mesenchymal cells and in human bladder are therefore likely to represent a mixed population of cell types. Enzymatic dispersal of biopsy samples yielded a heterogeneous population of cells, the majority of which were spindle-shaped smooth muscle cells and a smaller number of branched cells which resembled ICC, previously investigated in guinea-pig tissues (Johnston et al, 2008). The human bladder contains populations of c-Kit-positive ICC in the lamina propria and the detrusor. These ICC are structurally associated with cholinergic nerves and are similarly distributed to the ICC of the guinea-pig bladder (Davidson and McCloskey, 2005).