Following smooth muscle excitation and contraction, depletion of intracellular Ca²⁺ (Ca²⁺_i) stores activates capacitative Ca²⁺ entry (CCE) to replenish stores and sustain cytoplasmic Ca²⁺ elevations. The objectives of the present study were to characterize CCE and the Ca²⁺_i dynamics underlying human colonic smooth muscle contraction using tension recordings, fluorescent Ca²⁺_i indicator dyes and patch-clamp electrophysiology. The neurotransmitter acetylcholine (ACh) contracted tissue strips and, in freshly isolated colonic smooth muscle cells (SMCs), caused elevation of Ca²⁺_i as well as activation of non-selective cation currents. To deplete Ca²⁺_i stores, the sarcoplasmic reticulum Ca²⁺ -ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid were added to a Ca²⁺-free bathing solution. Under these conditions, addition of extracellular Ca²⁺ (3 mM) elicited increased tension that was inhibited by the cation channel blockers SKF-96365 (10 µM) and Lanthanum (100 µM) - suggestive of CCE. In a separate series of experiments on isolated SMCs, SERCA inhibition generated a gradual and sustained inward current. When combined with high-speed Ca²⁺ imaging techniques, CCE-evoked rise of Ca²⁺_i was associated with inward currents carrying Ca²⁺ that were inhibited by SKF-96365. Regional specializations in Ca²⁺ influx and handling during CCE were observed. Distinct 'hotspot' regions of Ca²⁺ rise and plateau were evident in 70% of cells, a feature not previously recognized in smooth muscle. We propose that store-operated Ca²⁺ entry occurs in 'hotspots' contributing to localized Ca²⁺ elevations of human colonic smooth muscle.