Conditioning prepulse of biphasic defibrillator waveforms enhances refractoriness to fibrillation wavefronts. The mechanism of biphasic waveform defibrillation threshold reduction is unknown. We tested the hypothesis that, during refractory period stimulation, sarcolemmal hyperpolarization by the first pulse of biphasic waveforms facilitates excitation channel recovery, which enhances graded responses produced by the second depolarizing pulse. This prolongs cellular refractoriness to fibrillation wavefronts when compared with a monophasic depolarizing stimulus. Monophasic (10 msec, rectangular wave) or symmetrical biphasic (10 msec, each pulse) current injection S2 stimuli at 1.5 and two times S1 threshold were used to scan the S1 action potential refractory period (S1 cycle length, 600 msec) in myocardial cell aggregates. S2 waveforms were delivered with normal and reversed polarity to test the hyperpolarizing action of biphasic waveforms. Responses to an S3 stimulus, which simulated a potential incoming fibrillation wavefront, were also determined. Results showed that biphasic S2 waveforms produced longer graded responses during and immediately after the S1 refractory period than did corresponding monophasic S2 waveforms. The maximum difference in response duration produced by the biphasic and monophasic waveforms was 58.6 +/- 10.0 msec (p less than 0.001). This maximum difference occurred 10 msec before the end of the S1 refractory period. The longer response durations produced by biphasic S2 also produced longer refractoriness to the S3 stimulus. The maximum difference in total refractoriness to S3 of 51.8 +/- 2.8 msec (p less than 0.002) occurred at the same S1S2 coupling interval as the maximum difference in S2 response duration. Prolonged refractoriness may protect ventricular cells from refibrillation wavefronts and act as the cellular basis for greater biphasic waveform defibrillation efficacy. 