Repolarization alternans has been considered a strong marker of electrical instability. The objective of this study was to investigate the hypothesis that ischemia-induced contrasting effects on the kinetics of membrane voltage and intracellular calcium transient (Ca_iT) can explain the vulnerability of the ischemic heart to repolarization alternans. Ischemia-induced changes in action potential (AP) and Ca_iT resulting in alternans were investigated in perfused Langendorff guinea pig hearts subjected to 10–15 min of global no-flow ischemia followed by 10–15 min of reperfusion. The heart was stained with 100 μl of rhod-2 AM and 25 μl of RH-237, and AP and Ca_iT were simultaneously recorded with an optical mapping system of two 16 × 16 photodiode arrays. Ischemia was associated with shortening of AP duration (D) but delayed upstroke, broadening of peak, and slowed decay of Ca_iT resulting in a significant increase of Ca_iT-D. The changes in APD were spatially heterogeneous in contrast to a more spatially homogeneous lengthening of Ca_iT-D. Ca_iT alternans could be consistently induced with the introduction of a shorter cycle when the upstroke of the AP occurred before complete relaxation of the previous Ca_iT and generated a reduced Ca_iT. However, alternans of Ca_iT was not necessarily associated with alternans of APD, and this was correlated with the degree of spatially heterogeneous shortening of APD. Sites with less shortening of APD developed alternans of both Ca_iT and APD, whereas sites with greater shortening of APD could develop a similar degree of Ca_iT alternans but slight or no APD alternans. This resulted in significant spatial dispersion of APD. The study shows that the contrasting effects of ischemia on the duration of AP and Ca_iT and, in particular, on their spatial distribution explain the vulnerability of ischemic heart to alternans and the increased dispersion of repolarization during alternans.