Impaired smooth muscle contractility is a hallmark of acute acalculous cholecystitis. While free cytosolic Ca²⁺ ([Ca²⁺]_i) is a critical step in smooth muscle contraction, possible alterations in Ca²⁺ homeostasis by cholecystitis have not been elucidated. Our aim was to elucidate changes in the Ca²⁺ signalling pathways induced by this gallbladder dysfunction. [Ca²⁺]_i was determined by epifluorescence microscopy in fura- 2 loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. F-actin content was quantified by confocal microscopy. Ca²⁺ responses to the InsP3 mobilizing agonist, CCK and to caffeine, an activator of the ryanodine receptors, were impaired in cholecystitic cells. This impairment was not due to a decrease in the size of the releasable pool. Inflammation also inhibited Ca²⁺ influx through L-type Ca²⁺ channels, as well as capacitative calcium entry induced by depletion of intracellular Ca²⁺ pools. In addition, the pharmacological phenotype of these channels was altered in cholecystitic cells. Inflammation impaired contractility further than Ca²⁺ signal attenuation, which could be related to the decrease in F actin that was detected in cholecystitic smooth muscle cells. These findings indicate that cholecystitis decreases both Ca²⁺ release and Ca²⁺ influx in gallbladder smooth muscle, but a loss in the sensitivity of the contractile machinery to Ca²⁺ may also be responsible for the impairment in gallbladder contractility.