Imaging of membrane potential (Vm) and intracellular calcium (Cai2+) is important for studying mechanisms of cardiac excitation, arrhythmias and defibrillation. We have developed an optical technique for simultaneous mapping of Vm and Cai2+ in cultured cell monolayers using fluorescent Vm- and Ca2+-sensitive dyes. Cultures of neonatal rat myocytes were double-stained with dyes RH-237 (Vm) and an analog of Fluo-3 or Rhod-2 dyes (Ca2+). These dyes have overlapping excitation spectra, allowing simultaneous excitation at the same wavelength range, and separate emission spectra allowing division of the fluorescent light into components sensitive to Vm and Cai2+, which were measured with two 16 x 16 photodiode arrays. It was found that Cai2+ measurements were strongly dependent on the properties of Ca 2+-sensitive dyes. Thus, high-affinity Ca2+ dyes such as Fluo-4 and Rhod-2 reported Cai2+ transients approximately twice as long as those reported by low-affinity dyes Fluo-4FF and Rhod-FF. In addition, dyes with different affinities resulted in different measurements of Cai2+ responses to electrical shocks. When shocks were applied during the early plateau phase of the action potential, low-affinity dyes reported transient Cai2+ decreases at sites of both negative and positive Vm changes. In contrast, high-affinity Ca 2+ dyes reported only a negligible change of plateau Ca i2+ and a large elevation of diastolic Cai2+. These discrepancies between high- and low-affinity dyes were explained by a model of dye-ion interaction, which indicated that apparently long Cai2+ transients and Cai2+ responses to electrical shocks measured with high-affinity dyes were due to their non-linear response. These results indicate that optical measurements of Cai2+ transient duration and shock-induced Ca i2+ changes require the use of low-affinity Ca i2+ dyes. © 2005 Elsevier Inc. All rights reserved.