We developed a new method for ratiometric optical mapping of transmembrane potential (Vm) in cardiac preparations stained with di-4-ANEPPS. V m -dependent shifts of excitation and emission spectra establish two excitation bands (<481 and 481nm) that produce fluorescence changes of opposite polarity within a single emission band (575-620nm). The ratio of these positive and negative fluorescence signals (excitation ratiometry) increases Vm sensitivity and removes artifacts common to both signals. We pulsed blue (450 ± 10nm) and cyan (505 ± 15nm) light emitting diodes (LEDs) at 375Hz in alternating phase synchronized to a camera (750 frames-per-second). Fluorescence was bandpass filtered (585 ± 20nm). This produced signals with upright (blue) and inverted (cyan) action potentials (APs) interleaved in sequential frames. In four whole swine hearts with motion chemically arrested, fractional fluorescence for blue, cyan, and ratio signals was 1.2 ± 0.3%, 1.2 ± 0.3%, and 2.4 ± 0.6%, respectively. Signal-to-noise ratios were 4.3 ± 1.4%, 4.0 ± 1.2%, and 5.8 ± 1.9%, respectively. After washing out the electromechanical uncoupling agent, we characterized motion artifact by cross-correlating blue, cyan, and ratio signals with a signal with normal AP morphology. Ratiometry improved cross-correlation coefficients from 0.50 ± 0.48% to 0.81 ± 0.25%, but did not cancel all motion artifacts. These findings demonstrate the feasibility of pulsed LED excitation ratiometry in myocardium. © 2006 IEEE.