Introduction: Fast spatially resolved measurements of transmembrane potential (V(m)) and intracellular calcium (Ca(i)2+) are important for studying mechanisms of arrhythmias and defibrillation. The goals of this work were (1) to develop an optical technique for simultaneous multisite optical recordings of V(m) and Ca(i)2+, and (2) to determine the relationship between V(m) and Ca(i)2+ during normal impulse propagation in myocyte cultures. Methods and Results: Monolayers of neonatal rat myocytes were stained with fluorescent dye RH-237 (V(m)) and Fluo-3AM (Ca(i)2+). Both dyes were excited at the same wavelength range. The emitted fluorescence was optically separated into components corresponding to changes in V(m) and Ca(i)2+ and measured using two 16 x 16 photodiode arrays at a spatial resolution of up to 27.5 μm per diode and sampling rate of 2.5 kHz. The optical setup was adjusted so that there was no optical cross-talk between the two types of measurements, which was validated in experiments involving staining with either RH-237 or Fluo-3. The amplitude of Fluo-3 signals rapidly decreased during experiments due to dye leakage. Dye leakage was substantially reduced by application of 1 mM probenecid, a blocker of organic anion transport, which had no effect on action potential duration and only minor effect on conduction velocity. In double-stained preparations, during regular pacing Ca(i)2+ transients had a rise time of 14.2 ± 2 msec, and they followed V(m) upstrokes with a delay of 5.3 ± 1 msec (n = 9). Durations of V(m) and Ca(i)2+ transients determined at 50% level of signal recovery were 54.6 ± 10 msec and 136 ± 8 msec, respectively. Application of 2 μM nifedipine reduced the amplitude and duration of Ca(i)2+ transients without significantly affecting conduction velocity. Conclusion: The results demonstrate feasibility of simultaneous optical recordings of V(m) and Ca(i)2+ transients with high spatial and temporal resolution.