Intracellular Ca²⁺ waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) wholemount preparations with intact mucosal layer (fullthickness, FT) by laser confocal imaging of intracellular Ca²⁺ and voltage recordings with microelectrodes, respectively. Spontaneous Ca²⁺ waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca²⁺ -induced Ca²⁺ release involving Ins(1,4,5)IP₃ receptors, and were not affected by tetrodotoxin and atropine. Spontaneous Ca²⁺ waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca²⁺ waves were abolished by the Ins(1,4,5)IP₃ receptor inhibitors, 2-APB and xestospongin C, and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. Acetylcholine, cholecystokinin, and carbachol augmented Ca²⁺ waves, and induced Ca²⁺ flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca²⁺ waves depend on sarco(endo)plasmic reticulum (SR) Ca²⁺ release via Ins(1,4,5)IP₃ receptors, PLC activity, Ca²⁺ influx via VDCCs, and SR Ca²⁺ concentration. Neurohormonal enhancement of GBSM excitability involves PLC dependent augmentation and synchronization of SR Ca²⁺ release via Ins(1,4,5)IP₃ receptors. Ca²⁺ waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.