The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca²⁺]_itransients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca²⁺]_itransients were recorded at depths ≤100 μm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked [Ca²⁺]_i transients were highly synchronized among neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50% (t _90–50%) and from 50 to 10% (t _50–10%) of the peak [Ca²⁺]_i were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2 Hz, 62 ± 3 and 94 ± 6 ms (n = 19, P < 0.05 vs. 1 Hz) in rhod-2-loaded cardiomyocytes. [Ca²⁺]_i decay was markedly slower in fura-2-loaded hearts (t _90–50% at 1 Hz, 128 ± 9 ms and at 2 Hz, 88 ± 5 ms;t _50–10% at 1 Hz, 214 ± 18 ms and at 2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of [Ca²⁺]_i decline resulted from increased cytosolic Ca²⁺ buffering, because the kinetics of rhod-2 decay resembled those obtained with fura-2 after incorporation of the Ca²⁺ chelator BAPTA. Propagating calcium waves and [Ca²⁺]_i amplitude alternans were readily detected in paced hearts. This approach should be of general utility to monitor the consequences of genetic and/or functional heterogeneity in cellular calcium signaling within whole mouse hearts at tissue depths that have been inaccessible to single-photon imaging.