Pulsed radiofrequency, in which short bursts of radiofrequency energy are applied to nervous tissue, has been recently described as an alternative technique devoid of nerve injury, a subsequent side effect of thermal lesions created by continuous radiofrequency lesioning. Yet the mechanism of this effect remains unclear. In this study we compared the acute effects of pulsed versus continuous radiofrequency energy on impulse propagation and synaptic transmission in hippocampal slice cultures and on cell survival in cortical cultures. A differential effect was observed on both systems, with pulsed radiofrequency producing a transient and continuous radiofrequency a lasting inhibition of evoked synaptic activity. In addition, although both continuous radiofrequency and pulsed radiofrequency treatments induced a distance-dependent tissue destruction under the stimulating needle, the effect was more pronounced in the continuous radiofrequency group. These findings suggest that the acute effects of pulsed radiofrequency are more reversible and less destructive in nature than the classic continuous radiofrequency mode, even in normothermal conditions. This model might help elucidate the importance of various parameters for the clinical application of radiofrequency lesioning and might open new horizons for the role of pulsed radiofrequency lesioning in cases of neuropathic pain.