The influence of iontophoresis and other physical enhancement methods such as electroporation and erbium:yttrium-aluminum-garnet (YAG) laser on the skin permeation of 5-fluorouracil (5-FU) was examined. Iontophoresis increased the in vitro transdermal transport of both the anionic and non-ionic forms of 5-FU. A combination of electroporation pretreatment and subsequent iontophoresis resulted in a higher permeation of 5-FU than either technique alone. It appeared that electroporation treatment exerted a disruptive influence on the stratum corneum (SC). The SC layers in the skin were partly ablated by the laser, resulting in a great enhancement effect on the skin permeation of 5-FU. Application of iontophoresis further increased the drug permeation across laser-pretreated skin. The laser was consistently the most potent technique to enhance 5-FU delivery among the physical enhancement methods examined in this study.