Gene transfer using electrical pulses is a rapidly expanding field. Many studies have been performed in vitro to elucidate the mechanism of DNA electrotransfer. In vivo, the use of efficient procedures for DNA electrotransfer in tissues is recent, and the question of the implied mechanisms is largely open. We have evaluated the effects of various combinations of square wave electric pulses of variable field strength and duration, on cell permeabilization and on DNA transfection in the skeletal muscle in vivo. One high voltage pulse of 800 V/cm, 0.1 ms duration (short high pulse) or a series of four low voltage pulses of 80 V/cm, 83 ms duration (long low pulses) slightly amplified transfection efficacy, while no significant permeabilization was detected using the 51Cr-EDTA uptake test. By contrast, the combination of one short high pulse followed by four long low pulses led to optimal gene transfer efficiency, while inducing muscle fibers permeabilization. These results are consistent with additive effects of electropermeabilization and DNA electrophoresis on electrotransfer efficiency. Finally, the described new combination, as compared to the previously reported use of repeated identical pulses of intermediate voltage, leads to similar gene transfer efficiency, while causing less permeabilization and thus being likely less deleterious. Thus, combination of pulses of various strengths and durations is a new procedure for skeletal muscle gene transfer that may represents a clear improvement in view of further clinical development. Copyright (C) 2000 Elsevier Science B.V.