Background - Optical mapping of cytosolic calcium transients in intact mammalian hearts is now possible using long-wavelength [Ca2+]i indicators. We propose that beat-to-beat [Ca2+]i transient alternans during ischemia may lead to spatial and temporal heterogeneity of calcium-activated membrane currents. Methods and Results - To test this hypothesis, isolated rabbit hearts were loaded with the fluorescent [Ca2+]i indicator, rhod-2 AM, and imaged at 300 frames/sec during blood-perfused ischemic trials. High-quality [Ca2+]i transients were recorded in each of 8 hearts. [Ca2+]i transient alternans was never present in control records but occurred in each of the hearts during ischemia, with onset after 2 to 4 minutes. Alternans was confined to circumscribed regions of the heart surface 5 to 15 mm across. Multiple regions of alternans were found in most hearts, and regions that were out of phase with one another were found in 6 hearts. Quantitative maps of alternans were constructed by calculating an alternans ratio. This ratio behaved as a continuous variable that reached a maximum value in the center of the regions with alternans. Conclusions - These results demonstrate marked spatial heterogeneity of the [Ca2+]i transient during the early phase of ischemia, which could produce electrical instability and arrhythmias in large mammalian hearts.