Caffeine-activated, large-conductance, non-selective cation channels (LCCs) have been found in the plasma membrane of isolated cardiomyocytes in several species. Yet, little is known about the effects of opening these channels. To examine such effects and to further understand the caffeine-activation mechanism, we carried out studies using whole-cell patch-clamp techniques with freshly isolated cardiomyocytes from rats and mice. Unlike previous studies, thapsigargin was used so that both the effect of opening LCCs and the action of caffeine were independent of Ca²⁺ release from intracellular stores. These Ca²⁺-permeable LCCs were found in a majority of the cells from both atria and ventricles, with a conductance of about 370 pS in rat atria. Caffeine and all of its direct metabolic products (theophylline, theobromine and paraxanthine) activated the channel, while isocaffeine did not. Although sharing some similarities with ryanodine receptors (RyRs, whose openings give rise to Ca²⁺ sparks), LCCs also showed some different characteristics. With simultaneous Ca²⁺ imaging and current recording, the localized fluorescence increase due to Ca²⁺ entry through a single opening of a LCC (SCCaFT) was detected. When membrane potential was recorded instead of current, SCCaFT-like fluorescence transients (indicating single LCC openings) were found to accompany membrane depolarizations. To our knowledge, this is the first report directly linking membrane potential changes to a single opening of an ion channel. Moreover, these events in cardiac cells suggest a possible additional mechanism by which caffeine and theophylline contribute to the generation of cardiac arrhythmias.