Although it has been suggested that multisite ventricular pacing alleviates heart failure by restoring ventricular electrical synchronization, the respective roles of voltage output, interventricular delay, and pacing sites in the development of ventricular arrhythmias occurrence have not been studied during biventricular pacing or LV pacing. Voltage-sensitive dye was used in eight ischemic Langerdorff-perfused guinea pig hearts to measure ventricular activation times and examine conduction patterns during multisite pacing from three RV and four LV sites. The hearts were stained with di-4-ANEPPS and mapped with a 16 x 16 photodiode array at a resolution of 625 microns per diode. Isochronal maps of RV and LV activation were plotted. Ischemia was produced by gradually halving the perfusion output over 5 minutes. Pacing the RV apex and the base of the LV anterior wall was associated with the most homogeneous and rapid activation pattern (28 +/- 9 vs 41 +/- 12 ms with the other configurations, P < 0.01), and no inducible arrhythmia. In six hearts, ventricular tachycardia could be induced when pacing from the right and left free walls with 20 ms of interventricular delay, at six times the pacing threshold output. In four hearts, simultaneous RV and LV pacing at high voltage output induced ventricular fibrillation with complex three-dimensional propagation patterns, independently of the pacing sites. During biventricular pacing with ischemia, pacing at high voltage output with a long interventricular delay is likely to induce ventricular arrhythmias, particularly when left and right pacing results in a conduction pattern orthogonal to the ventricular myocardial fibers orientation.