This study investigates the involvement of capacitative Ca 2+ entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM–1 μM, a sarcoplasmic reticulum Ca 2+-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 μM) after 55 min. Contractile responses to thapsigargin (1 μM) were abolished in a Ca 2+-free medium. Subsequent re-addition of Ca 2+ (2.5 mM) produced a sustained tonic contraction (99 ± 6% of the carbachol response). The contractile response to Ca 2+ re-addition following incubation of tissues in a Ca 2+-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 ± 4 % vs 100 ± 7 % of carbachol; p < 0.05). Contractile responses to Ca 2+ re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca 2+ channel antagonist, nifedipine (10 μM) and (b) the general inhibitor of Ca 2+ entry channels including store-operated channels, SK&F96365 (50 μM and 100 μM). In separate experiments, responses to Ca 2+ re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 μM). These results suggest that capacitative Ca 2+ entry provides a source of activator Ca 2+ for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca 2+ re-addition following depletion of sarcoplasmic reticulum Ca 2+ stores with thapsigargin, are mediated in part by Ca 2+ entry through voltage-operated Ca 2+ channels and by capacitative Ca 2+ entry through store-operated Ca 2+ channels which can be blocked by SK&F96365. Furthermore, capacitative Ca 2+ entry in this tissue may be modulated by tyrosine kinase.