Recently, myofibroblastic cells have been identified in the lamina propria of the human and other species. Those cells form a distinct layer underneath the L-Asarinin urothelium in close proximity to afferent nerves and we therefore refer to these cells as suburothelial myofibroblasts. There is an ongoing debate as to whether these cells are indeed interstitial cells of Cajal as promoted by McCloskey in a recent review. However, while c-kit positive cells resembling ICCs are numerous in guinea-pig and pig bladders only a subpopulation of vimentin and alpha-smooth muscle cell actin positive cells also stain positive for c-kit. Since the physiological ATP concentration during the initial filling phase is likely to be very low, we hypothesized that the Ca2+ activity of the sMF is affected at very low ATP concentrations. Furthermore, it was hypothesized that the spontaneous activity of the sMF is likely to be connected with the generation or amplification of the afferent signals. Thus the autonomous activity of the detrusor by sMF activity. In the present study we investigated the ATP induced modulation of spontaneous activity, intracellular calcium response, and purinergic signaling in cultured human suburothelial myofibroblasts. The growing cells showed typical morphological and immunohistochemical features of myofibroblasts as recently described. The use of special smooth muscle cell growth medium was sufficient to avoid growth of urothelial cells as proven by visual phase contrast and lack of cells staining positive for cytokeratin. For calcium imaging experiments cells were plated onto 13mm glass coverslips coated with collagen A and grown to a confluence of about 80% for the calcium imaging experiments. Alterations of spontaneous contractile activity of the bladder have been described in bladder strips from patients with Paederosidic-acid-methyl-ester idiopathic detrusor instability and from patients with neurogenic detrusor overactivity. In the latter, spontaneous contractile activity was not altered by removing the urothelium/suburothelium from the bladder strips which indicates that the spontaneous detrusor contractions were initialized within the detrusor itself.