There is an increasing body of evidence that the urinary bladder contains, in addition to bulk smooth muscle, a population of cells with many characteristics of interstitial cells. These cells can be identified using antibodies to the Kit receptor both in fixed tissue preparations and when enzymatically dispersed [1]. The physiological roles of bladder interstitial cells has yet to be established; however, information on their electrophysiological and Ca²⁺ signalling characteristics is beginning to emerge [2-4]. The major excitatory innervation to the bladder is cholinergic and it was therefore of interest to investigate the response of freshly-dispersed interstitial cells to cholinergic stimulation. Bladders were removed post mortem from guinea-pigs of either sex; they were opened longitudinally and the mucosa removed. Cells were isolated from the detrusor region as previously described (McCloskey & Gurney, 2002), loaded with 1μM Fluo 4AM and studied using confocal microscopy. Application of 1 or 10μM carbachol induced an increase in intracellular Ca²⁺. These responses were repeatable after 70s and comprised large transients lasting several seconds. The carbachol-induced transients were not blocked by 1μM nifedipine (n=7). The participation of Ca²⁺ release from intracellular stores was initially investigated using 2-APB, often used to block release from IP3-sensitive stores. The effect with this drug on the carbachol responses was variable with no effect seen in 7 cells and a reduction seen in 4 others; however, xestospongin-C, regarded as a better blocker of IP₃ receptors did block the responses (n=6) suggesting that release of Ca²⁺ from IP₃-sensitive stores was a major component of the Ca²⁺ transients. Previous work had shown that the carbachol responses were sensitive to the muscarinic blocker atropine, so pharmacological blockers of the M2 and M3 receptors were used to examine this further. Methoctramine, an M2 receptor antagonist had little effect on the carbachol responses (n=4) whereas the M3 receptor antagonist, 4-DAMP inhibited the responses (n=2). In summary, detrusor interstitial cells respond to cholinergic stimulation by firing Ca²⁺ transients. These appear to be mediated via M3 muscarinic receptors as they were blocked by atropine and 4-DAMP but not methoctramine. A major source of Ca²⁺ appears to be via the IP₃-sensitive store as the responses were sensitive to xestospongin-C but not nifedipine. The findings of this study suggest that detrusor interstitial cells could participate in cholinergic neurotransmission in the normal bladder.