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 ΔVm 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 ΔV+m was caused by the outward flow of ICaL and troponin binding; at sites of ΔV−m 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 ΔV+m 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 ΔCai2+ 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 ΔV−m and ΔV+m. 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 Cai2+, likely caused by membrane electroporation.