Current techniques to map intramural activation patterns in ex vivo cardiac tissue have limited spatial resolution. Here, we report on the experimental validation of a novel optical technique that was recently proposed to resolve the size and depth of intramural wave fronts using alternating transillumination (AT). AT was achieved by simultaneously mapping the epi- and endocardial surfaces with two synchronized CCD cameras and rapidly alternating LED illumination between both surfaces. Optical phantoms were made based on tissue optical properties measured using a hybrid optical spectrometer. Spherical fluorescent sources (Scarlet microspheres, Invitrogen, UK) of varying sizes were embedded at known depths in the phantoms. Coronary-perfused procine left ventricular slab preparations were stained with DI-4-ANBDQBS (n = 3) and paced at known intramural depths. In phantoms we were able to reliably estimate the depth of the center of fluorescent sources (9.6 ± 5.4% error), as well as their transmural extent (15.7 ± 11.5% error). In ventricular slabs we were able to localize the sites of origin of intramural excitation waves with a precision of ± 1.6 mm. Transmural conduction velocities were, for the first time, measured optically from the surfaces and found to be 21.0 ± 12.4 cm/s. In conclusion, alternating transillumination is a promising technique for reliable reconstruction of depth and expansion rate of intramural activation wave fronts in cardiac tissue.