The present study examined pacing site-dependent changes in QT interval and transmural dispersion of repolarization (TDR) and their potential role in the development of torsade de pointes (TdP). In humans, the QT interval, JT interval, and TDR were measured in 29 patients with heart failure during right ventricular endocardial pacing (RVEndoP), biventricular pacing (BiVP), and left ventricular epicardial pacing (LVEpiP). In animal experiments, pacing site--dependent changes in ventricular repolarization were examined with a rabbit left ventricular wedge preparation in which action potentials from endocardium and epicardium could be simultaneously recorded with a transmural ECG. In humans, LVEpiP and BiVP led to significant QT and JT prolongation. LVEpiP also enhanced TDR. Frequent R-on-T extrasystoles generated by BiVP and LVEpiP but completely inhibited by RVEndoP occurred in 4 patients, of whom 1 developed multiple episodes of nonsustained polymorphic ventricular tachycardia and another suffered incessant TdP. In rabbit experiments, switching from endocardial to epicardial pacing produced a net increase in QT interval and TDR by 17+/-5 and 22+/-5 ms, respectively (n=6, P<0.01), without parallel increases in ventricular transmembrane action potential durations. Epicardial pacing facilitated transmural propagation of early afterdepolarization, leading to the development of R-on-T extrasystoles and TdP in the presence of action potential duration-prolonging agents. LVEpiP and BiVP increase QT, JT, and TDR by altering the transmural sequence of activation of the intrinsically heterogeneous ventricular myocardium. Our data suggest that the resultant exaggeration of arrhythmic substrates can lead to the development of TdP in a subset of patients.