Changes in intracellular calcium concentration (ΔCai2+) induced by electrical shocks may play an important role in defibrillation, but high-resolution ΔCai2+ measurements in a multicellular cardiac tissue and their relationship to corresponding Vm changes (ΔVm) are lacking. Here, we measured shock-induced ΔCai2+ and ΔV m in geometrically defined myocyte cultures. Cell strands (width=0.8 mm) were double-stained with Vm-sensitive dye RH-237 and a low-affinity Cai2+-sensitive dye Fluo-4FF. Shocks (E≈5 to 40 V/cm) were applied during the action potential plateau. Shocks caused transient Cai2+ decrease at sites of both negative and positive ΔVm. Similar Cai2+ changes were observed in an ionic model of adult rat myocytes. Simulations showed that the Cai2+ decrease at sites of ΔVm+, was caused by the outward flow of ICaL and troponin binding; at sites of ΔVm- it was caused by inactivation of ICaL combined with extrusion by Na-Ca exchanger and troponin binding. The important role of ICaL was supported by experiments in which application of nifedipine eliminated Cai2+ decrease at ΔVm+. sites. Largest ΔCai2+ were observed during shocks of ≈10 V/cm causing simple monophasic ΔVm. Shocks stronger than ≈20 V/cm caused smaller ΔCai2+ and postshock elevation of diastolic Cai2+. This was paralleled with occurrence of biphasic negative ΔVm that indicated membrane electroporation. Thus, these data indicate that shocks transiently decrease Cai2+ at sites of both ΔVm- and ΔVm+. Outward flow of ICaL plays an important role in Cai2+ decrease in the ΔV m+ areas. Very strong shocks caused smaller negative ΔCai2+ and postshock elevation of diastolic Ca i2+, likely caused by membrane electroporation.