Simultaneous mapping of transmembrane voltage (V(m)) and intracellular Ca(2+) concentration (Ca(i)) has been used for studies of normal and abnormal impulse propagation in cardiac tissues. Existing dual mapping systems typically utilize one excitation and two emission bandwidths, requiring two photodetectors with precise pixel registration. In this study we describe a novel, single-detector mapping system that utilizes two excitation and one emission band for the simultaneous recording of action potentials and calcium transients in monolayers of neonatal rat cardiomyocytes. Cells stained with the Ca(2+)-sensitive dye X-Rhod-1 and the voltage-sensitive dye Di-4-ANEPPS were illuminated by a programmable, multicolor LED matrix. Blue and green LED pulses were flashed 180° out of phase at a rate of 488.3 Hz using a custom-built dual bandpass excitation filter that transmitted blue (482 ± 6 nm) and green (577 ± 31 nm) light. A long-pass emission filter (>605 nm) and a 504-channel photodiode array were used to record combined signals from cardiomyocytes. Green excitation yielded Ca(i) transients without significant crosstalk from V(m). Crosstalk present in V(m) signals obtained with blue excitation was removed by subtracting an appropriately scaled version of the Ca(i) transient. This method was applied to study delay between onsets of action potentials and Ca(i) transients in anisotropic cardiac monolayers.