In vivo gene transfer to muscle tissue by electroporation can produce long-term, high-level gene expression. In the present study, we report quantitative results on muscle fiber permeabilization using Cr(51)-EDTA as a marker, and we analyze the influence of electric field strength, pulse duration, and pulse number. The comparison of these results to recently published data on gene transfer (Mir et al., P.N.A.S. (USA), 1999), using an identical experimental setup provides the basis for discussing the importance of the level of permeabilization for gene transfer. The threshold for permeabilization was determined by measuring uptake of Cr(51)-EDTA, and DNA transfer was optimal for field strengths just above the threshold for permeabilization. This means that when designing in vivo electric-field-mediated gene transfer protocols for various tissues, determining the threshold for permeabilization using a rapid test such as incorporation of Cr(51)-EDTA, can be used to predict the optimal window for gene transfer.