The influence of activation sequences on action potential configuration, especially in the repolarization phase, was examined in isolated canine ventricular muscles. Action potentials were recorded from the epicardial surface in the center of a preparation having nearly uniform fiber orientation (25 X 25 mm). Stimuli applied just adjacent to the recording site produced nearly centrifugal propagation. An activation sequence either parallel (longitudinal) or perpendicular (transverse) to the long axis of the muscle fibers was produced by peripheral stimulation. Action potential duration at -60 mV (APD-60 mV) during centrifugal propagation was significantly longer than that during longitudinal propagation. Further shortening of APD-60 mV was observed during transverse propagation. When a collision of longitudinal or transverse wavefronts (longitudinal or transverse collision) was produced at the action potential recording site, the shortest APD was recorded. During centrifugal propagation, action potential mapping around the stimulating electrodes revealed that APD-60 mV shortened gradually as the recording site was moved further from the stimulation site. The spatial gradient of APD was steeper in the transverse than in the longitudinal direction, causing a distortion in the repolarization sequence and the recovery of excitability near the center of the tissue. Premature stimuli applied to an area near the central stimulation site induced one-way block and circus movement of the wavefront, indicating reentry of excitation. We concluded that the activation sequence and anisotropic cellular geometry substantially affect APD, and that such a change contributes to the spatial inhomogeneity of refractoriness leading to reentrant arrhythmias.