Interstitial cells of Cajal (ICC) act as pacemaker cells in the rabbit urethra to stimulate smooth muscle to contract and maintain myogenic tone during bladder filling (Sergeant et al., 2000). Pacemaker electrical activity in urethral ICC results from propagating Ca2+ waves that stimulate Ca2+ activated Cl- channels. The purpose of the present study was to examine the role of Ca2+ influx and diffusion in the initiation and propagation of the waves. All the experiments described were approved by DkIT Animal Care Committee. New Zealand white rabbits were humanely killed and strips of urethral smooth muscle were dissected and enzymatically dispersed to release single ICCs. These were allowed to settle on a glass bottomed dish, loaded with Fluo – 4 AM (500 nM) and imaged using a spinning disk confocal microscope. Cells were superfused with Hanks solution at 37oC. Under control conditions spontaneous increases in fluorescence (indicating calcium release from the endoplasmic reticulum, ER) occurred at various distances along the cell length. These varied in magnitude (from 1.3 to 14.0 F/F0, mean 5.5 ± SEM 0.2, n=146 events in 6 cells). The length of spread varied in a continuum from 1.6 µm in the case of transient events to 183 µm in the case of full propagating waves with a mean value of 17.6 ± 2.4. Sometimes waves arose simultaneously at opposite ends of the cell and when they collided this caused mutual annihilation. The development of propagating waves depended on three factors: influx of calcium from the extracellular medium; diffusion of calcium within the cell and the level of IP3 within the cell. Thus in a cell that was firing regular propagated waves, removal of extracellular calcium abolished these leaving only short-lived calcium transients which did not propagate. Similarly when diffusion of calcium within the cell was inhibited, by adding 3µM EGTA-AM to the external solution, propagated waves were blocked, leaving short-lived calcium transients. Conversely in cells that exhibited only transient calcium increases these events developed into propagated waves when agonists (such as phenylephrine, which increased intracellular IP3 levels) were added. Furthermore increasing the sensitivity of calcium release from ryanodine receptors (RyR) by adding 1mM caffeine to calcium-free external solution caused transient calcium events to develop into propagated waves. We conclude from these results that transient calcium events result from spontaneous release of Ca2+ from the ER. These can develop into propagated waves when sufficient cytoplasmic calcium and IP3 exists to cause sensitization of adjacent release sites. Wave propagation occurs when calcium diffuses to an adjacent sensitized release site and stimulates regenerative release.