The mechanism for the urinary bladder to sense fullness is unresolved. Evidence suggests that ATP release from urothelium and its eventual action on sub-urothelial sensory afferent purinoceptors may be involved, but the signal transduction process is far from clear (Ferguson et al 1997; Cockayne et al 2000). Recently we proposed a role for sub-urothelial myofibroblasts as an intermediate variable gain stage in bladder sensation and demonstrated that these cells possess many characteristics of excitable cells and respond to ATP (Wu et al 2003). One physiologically important feature of these cells was the presence of spontaneous spikes of membrane depolarisation. The objective of this study was to clarify, using guinea-pig preparations, the cellular basis of these spontaneous activities. Urinary bladders were obtained from guinea-pigs sacrificed by a schedule 1 method. Myofibroblasts were dissociated from the urothelium and identified by vimentin staining (Sui et al 2002). Experiments were performed in HCO₃ /CO₂ – buffered superfusate at 37°C, pH 7.4. Electrophysiological recordings were made with patch electrodes filled with a high K⁺ based intracellular medium. Intracellular Ca²⁺,[Ca²⁺]_i, was measured simultaneously by dialysing fluorochrome fura-2 via the patch pipette.Spontaneous inward currents were recorded in 26/58 cells held at a resting potential of −60mV. These inward currents were accompanied by a spontaneous rise of [Ca²⁺]_i. Phase-loop analysis showed that the rise of [Ca²⁺]_i preceded the inward currents. Variation of membrane potential altered the magnitude and polarity of spontaneous currents whilst the [Ca²⁺]_i rise was little affected. The reversal potential for the current was -26±8 mV (SD, n=8), close to the equilibrium potential of Cl^–ions under the experimental conditions. Suppression of the spontaneous [Ca²⁺]_i rise by Ca deprivation abolished the inward current (n=3). Raising [Ca²⁺]_i, either by thapsigargin (0.5µM, n=6) or ionomycin (10µM, n=3), evoked the membrane current. Spontaneous inward currents with associated Ca²⁺ rises could be triggered following application of ATP. These results show that spontaneous inward currents are present in sub-urothelial myofibroblasts to support spontaneous electrical activity. These currents are mainly Ca²⁺-activated Clcurrents and tightly regulated by spontaneous changes to [Ca²⁺]_i. Existence of these active, Ca²⁺ responsive currents provides a distinct coupling mechanism between [Ca²⁺]_i and membrane electrical activity, subject to regulation by the putative sensory mediator ATP.