Extracellular recording and stimulation techniques have been used to demonstrate that the effective refractory period of epicardial ventricular cells is significantly influenced by the sequence of activation. Whether myocardial fiber orientation is also important in determining the repolarization process is unclear. To determine the importance of fiber orientation on the repolarization process, we studied 12 blocks of pig right ventricular tissue in vitro. The size of each tissue block was 30 x 30 x 2 mm. Transmembrane action potentials were recorded, and effective refractory periods were measured from the preparation's epicardial surface, which showed nearly uniform fiber orientation. Tissues were paced at 500- and 1,000-ms cycle lengths. Sequential recordings were made at 1, 4, 7, 10, 13, and 16 mm from the stimulation site along and across the fibers. The results show that propagation of depolarization was much slower in the transverse direction than in the longitudinal direction. In the transverse direction, action potential duration was longest at the closest observation point, i.e., 1 mm from the stimulation, site (188 +/- 14 and 267 +/- 18 ms for 500- and 1,000-ms pacing cycle lengths, respectively). Action potential duration progressively shortened as the recording site was moved farther from the stimulation site (P < 0.001). The action potential duration 16 mm from the stimulation site was 165 +/- 11 and 247 +/- 12 ms for 500- and 1,000-ms pacing cycle lengths, respectively. In contrast, the action potential duration in the longitudinal direction did not change as the distance between the recording site and stimulation site increased. We conclude that, at physiological temperature and pacing cycle lengths, sequence of activation significantly influenced action potential duration when the propagation of activation was transverse to myocardial fiber orientation. When activation propagated parallel to fiber orientation, there was little or no change of action potential duration as distance increased.