Acetylcholine released from parasympathetic excitatory nerves activates contraction in detrusor smooth muscle. Immunohistochemical labeling of guinea-pig detrusor with anti-c-Kit and anti-VAChT demonstrated a close structural relationship between interstitial cells of Cajal (ICC) and cholinergic nerves. The ability of guinea-pig bladder detrusor ICC to respond to the acetylcholine analogue, carbachol, was investigated in enzymatically dissociated cells, loaded with the Ca²⁺-indicator fluo 4AM. ICC fired Ca²⁺-transients in response to stimulation by carbachol (1/10µM). Their pharmacology was consistent with carbachol-induced contractions in strips of detrusor which were inhibited by 4-DAMP (1µM) an M₃ receptor antagonist but not by the M₂ receptor antagonist methoctramine (1µM). The source of Ca²⁺ underlying the carbachol-transients in isolated ICC was investigated using agents to interfere with influx or release from intracellular stores. Nifedipine (1µM) or Ni²⁺ (30-100µM) to block Ca²⁺-channels or the removal of external Ca²⁺reduced the amplitude of the carbachol-transients. Application of ryanodine (30µM) or tetracaine (100µM) abolished the transients. The phospholipase C inhibitor, U-73122 (2.5µM) significantly reduced the responses. 2-APB (30µM) caused a significant reduction and Xestospongin C (1µM) was more effective, almost abolishing the responses. Intact in situ preparations of guinea-pig bladder loaded with a Ca²⁺-indicator showed distinctively different patterns of spontaneous Ca²⁺ events in smooth muscle cells and ICC. Both cell types responded to carbachol by an increase in frequency of these events. In conclusion, guinea-pig bladder detrusor ICC, both as isolated cells and within whole tissue preparations, respond to cholinergic stimulation by firing Ca²⁺-transients.