The properties of suburothelial myofibroblasts are described, and their possible role in shaping sensory responses from the bladder wall are discussed. Suburothelial myofibroblasts consist of long spindle-shaped cells that form a distinctive layer below the urothelium and are connected to each other through connexin 43 gap junctions. Isolated cells from guinea pig or human bladders display spontaneous fluctuations of membrane potential and intracellular [Ca2+], and respond in a similar way to exogenous application of adenosine triphosphate (ATP) or lowering of extracellular pH. ATP generates an intracellular Ca2+ transient via activation of a P2Y receptor, which in turn initiates a Ca2+-sensitive Cl− current inward at the normal membrane potential of −50 to −60 mV. Of the P2Y receptor subtypes identified by immunolabeling, the most prominent was the P2Y6 receptor. Cell pairs, without the formation of gap junctions, elicit augmented responses to exogenous agonists. Mechanical stimulation of the suburothelial layer in intact cross-sections of the bladder elicited Ca2+ waves that propagated across the suburothelial layer before invading the detrusor layer. This indicates that the suburothelial layer forms a discrete functional layer of cells capable of propagating signals over many cell lengths. A function for suburothelial myofibroblasts is proposed whereby they act as an amplification stage in the sensory response to bladder-wall stretch, as occurs during bladder filling. Neurourol. Urodynam. 26:914–919, 2007. © 2007 Wiley-Liss, Inc.