Bladder sub-urothelial myofibroblasts may modulate both sensory responses from the bladder wall and spontaneous activity. This study aimed to characterise further these cells in their response to exogenous agents implicated in mediating the above activity. Detrusor strips, with or without mucosa, and isolated sub-urothelial myofibroblasts were prepared from guinea-pig bladders. Isometric tension, intracellular Ca²⁺ and membrane current were recorded. Cell pairs were formed by pushing two cells together. Tension, intracellular Ca²⁺ and membrane potential were also recorded from bladder sheets using normal or spinal cord-transected (SCT) rats. Spontaneous contractions were greater in detrusor strips with an intact mucosa, and was augmented by 10 µM UTP. ATP, UTP or reduced extracellular pH elicited Ca²⁺ transients and inward currents (E_rev -30 mV) in isolated cells. Capsaicin (5-30 µM) reduced membrane current (37±12% of control) with minor effects on Ca2+ transients: Na nitroprusside reduced membrane currents (40±21% of control). Cell pair formation, without increase of cell capacitance, augmented ATP and pH responses (180±58% of control) and reduced the threshold to ATP and acidosis. Glivec (20-50 µM) reversibly blocked the augmentation and also reduced spontaneous activity in bladder sheets from SCT, but not normal, rats. Glivec also disrupted the spread of Ca²⁺ waves in SCT sheets generating patterns similar to normal bladders. Sub-urothelial myofibroblasts respond to exogenous agents implicated in modulating bladder sensory responses; responses augmented by physical intercellular contact. The action of glivec and its selective suppression of spontaneous activity in SCT rats identifies a possible pathway to attenuate bladder overactivity.