Background: Pulsed field ablation (PFA) is a novel nonthermal cardiac ablation technology based on irreversible electroporation. Unfortunately, the characteristics of the electric field waveforms used in clinical and experimental PFA are not typically reported. This study examines the effect of the frequency of biphasic waveforms and compares biphasic to monophasic waveforms. Methods: A total of 29 Sprague-Dawley rats were treated with PFA using an epicardial monopolar electrode. Biphasic waveforms with three different frequencies, 90, 260, and 450 kHz (10 bursts of 100 µs duration at 500 V or 800 V) and monophasic waveforms (10 pulses of 100 µs duration at 500 V) were studied. Collateral neuromuscular stimulation and temperature increase in the point of application were directly measured. Lesion formation was assessed 3 weeks after treatment by histopathologic analysis. Computer simulations were used to estimate the electric field lethal threshold for each condition. A previous in vitro study was performed to draw a complete characterization of the studied dependencies. Results: Morphometric analysis demonstrated a significant association between chronic lesion size and waveform characteristics. For the same voltage level, monophasic waveforms yielded the largest lesions compared with any of the biphasic protocols ( P <0.05). Increasing PFA frequency was associated with reduced neuromuscular stimulation but also with reduced ablation efficacy. Maximum absolute temperature increase recorded along a complete treatment was 3 °C. Vascular structures inside the lesions were preserved for all conditions. Computer simulation-based analysis showed that waveform frequency had a graded effect on the lethal electric field threshold, with threshold of 600 V/cm for monophasic waveforms versus 2000 V/cm for biphasic waveforms with a frequency of 450 kHz. Conclusions: Frequency is a major determinant of efficacy in biphasic PFA. Our results highlight the critical need of disclosing waveform characteristics when reporting the results of different PFA systems.