Monitoring action potential (AP) characteristics in intact tissue gives an insight into the electrical behaviour of cardiomyocytes in situ. Combining these measurements with intracellular Ca2+ ([Ca2+ ]i) measurements gives a better understanding of the relationship between voltage changes and Ca2+ flux. Previous studies have described voltage and Ca2+ measurements in intact hearts using widefield epifluorescence techniques. However, this does not provide the depth resolution required for investigating transmural heterogeneities in whole heart preparations. Two photon laser scanning microscopy (2PLSM) enables measurements to be made in multicellular preparations with subcellular depth resolution. This study describes the feasibility of measuring voltage and [Ca2+]i in whole hearts using 2PLSM for transmural investigations. Rabbit, rat and mouse hearts were Langendorff perfused and contraction inhibited with 10mM butanedione monoxime and 10µM blebbistatin. Hearts were electrically paced and positioned horizontally for imaging of the left ventricle (LV). Epicardial membrane potential recordings were made with sharp microelectrode (ME), resistance 50MΩ with 1M KCl for comparison with 2-photon (2P) measurements to validate the technique. Ratiometric voltage signals were measured using di-4-ANEPPS (excitation 920nm) and Ca2+ signals using fura-2 AM (excitation 760nm). Values are means±S.E.M, compared by paired t-test. Using 2PLSM, sequential voltage and Ca2+ recordings were initiated 50µm below the LV surface and continued to a maximum depth of approximately 500µm. Poor signal:noise prevented deeper measurements. Comparison of AP duration at 90% repolarisation (APD90) and upstroke rise time (TRise) obtained by ME and 2P methods revealed no difference in APD90 (134.4±4.7ms vs. 133.4±7.1ms, n=4 rabbit; 49.7±6.0ms vs. 51.6±6.8ms, n=3 rat and 50.7±4.1ms vs. 55.3±7.2ms, n=4 mouse). No difference in TRise was observed in rabbit APs (2.54±0.24ms ME vs. 2.88±0.30ms 2P). However, in rat and mouse hearts, TRise values obtained from ME measurements were faster than those obtained by 2P methods (0.50±0.05ms vs. 0.92±0.13ms in rat, p<0.05 and 0.65±0.06ms vs. 1.18±0.23ms in mouse, p=0.065). Subsampling of ME data from 28kHz to 2.8kHz to more closely match 2P sampling frequencies of 2.6kHz reduced the difference in TRise values (new rat ME TRise 0.70±0.02ms, new mouse ME TRise 0.78±0.03ms). 10-90% rise times of Ca2+ transients were slower (no faster than 5ms in all species), and could thus be accurately sampled with 2P measurements. In conclusion, voltage and Ca2+ sensitive dyes can be used with 2PLSM to study transmural differences in AP and Ca2+ signalling characteristics. This technique is best suited for use in rabbit hearts, where upstroke velocity is sufficiently slow to be faithfully recorded with 2P sampling frequencies.