Recent evidence suggests that ion channels governing the response of action potential duration (APD) to a premature stimulus (ie, APD restitution) are heterogeneously dispersed throughout the heart. However, because of limitations of conventional electrophysiological recording techniques, the effects of restitution in single cells on ventricular repolarization at the level of the intact heart are poorly understood. Using high-resolution optical mapping with voltage-sensitive dyes, we measured APD restitution kinetics at 128 simultaneous sites on the epicardial surface (1 cm2) of intact guinea pig hearts (n = 15). During steady state baseline pacing, APD gradients that produced a spatial dispersion of repolarization were observed. Mean APD was shortened monotonically from 186 +/- 19 ms during baseline pacing (S1-S1 cycle length, 393 +/- 19 ms) to 120 +/- 4 ms as single premature stimuli were introduced at progressively shorter coupling intervals (shortest S1-S2, 190 +/- 15 ms). In contrast, premature stimuli caused biphasic modulation of APD dispersion (defined as the variance of APD measured throughout the mapping field). Over a broad range of increasingly premature coupling intervals, APD dispersion decreased from 70 +/- 29 ms2 to a minimum of 10 +/- 7 ms2 at a critical S1-S2 interval (216 +/- 18 ms), and then, at shorter premature coupling intervals, APD dispersion increased sharply to 66 +/- 25 ms2. Modulation of APD dispersion by premature stimuli was attributed to coupling interval-dependent changes in the magnitude and direction of ventricular APD gradients, which, in turn, were explained by systematic heterogeneities of APD restitution across the epicardial surface. There was a characteristic pattern in the spatial distribution of cellular restitution such that faster restitution kinetics were closely associated with longer baseline APD. This relationship explained the reversal of APD between single cells, inversion of APD gradients across the heart, and ECG T-wave inversion during closely coupled premature stimulation. Therefore, because of the heterogeneous distribution of cellular restitution kinetics across the epicardial surface, a single premature stimulus profoundly altered the pattern and synchronization of ventricular repolarization in the intact ventricle. This response has important mechanistic implications in the initiation of arrhythmias that are dependent on dispersion of repolarization.