Interstitial cells (IC) have been shown to exist within the wall of the guinea-pig bladder (1). Previous work has shown that guinea-pig bladder IC display spontaneous Ca2+signals (1, 2) and isolated IC respond to cholinergic stimulation by firing Ca2+-transients. The aim of the present investigation was to study Ca2+-signalling in IC and smooth muscle cells in in vitro preparations. Bladders were removed from guinea-pigs, killed by cervical dislocation in accordance with Schedule 1 UK Home Office regulations. Small preparations, containing a few smooth muscle bundles were loaded with fluo-4AM and imaged using a confocal microscope. After washing with Krebs to remove excess indicator, areas of the tissue in which spontaneous Ca2+ rises occurred were imaged with the confocal microscope. Two types of activity were typically present; Ca2+-waves in smooth muscle cells (see below) and integrated, co-ordinated flashes in the whole smooth muscle bundle (amplitude F/F0 0.61±0.07, duration 3.5±0.5sec, frequency 3.8±0.9 per minute, n=7). In addition, spontaneous Ca2+ transients were observed in the IC found on the edge of the bundles or in the interstitium between two or more bundles. The Ca2+-events in smooth muscle cells and IC were markedly different. The smooth muscle cells fired regular signals of 2.3±0.2s duration, amplitude 0.21±0.03 at 9.2±1.7 per minute (n=8) whereas the IC fired transients of duration 12.2±2.4s, amplitude 0.49±12, and less frequently at 1.3±0.3/min. Application of 10µM nifedipine abolished the co-ordinated whole bundle flashes but did not prevent Ca2+-waves in individual smooth muscle cells (at 6.1±1.6/min control to 4.9±1.0/min in drug, n=4) nor the transients in IC (from 1.91/min to 0.79/min in nifedipine, n=4). Activity of the cells was enhanced by depolarizing the tissue with 60mM K+ external solution. Smooth muscle cells and IC both responded with an initial large increase in intracellular Ca2+ followed by oscillatory events. This was often accompanied by tissue contraction. In smooth muscle cells, carbachol (30µM) increased frequency from 11±1/min to 21±2.9/min, n=5). Carbachol caused large Ca2+ transients (0.34±0.02 amplitude) in IC followed by several oscillations. In summary, spontaneous Ca2+-signalling occurs in both smooth muscle cells and IC in the guinea-pig bladder. The signals differ significantly between the two cell types. Co-ordination of global flashes in bundles relies on influx through voltage-dependent channels. Both cell types responded to cholinergic stimulation.