The aim of the present study was to evaluate the influence of the lipids EPC (L-alpha-phosphatidylcholine, egg lecithin), DSPC (distearoylphosphatidylcholine), and SA (stearylamine) on the iontophoretic mannitol transport through human skin in vitro. The skin was pretreated with 1 mM lipid suspension with ethanol (32%) for 24 h prior to the iontophoretic experiment with mannitol. In addition, the penetration of fluorescent lipids into the epidermis during the pretreatment was studied by confocal laser scanning microscopy (CLSM). The results of the present study show that pretreatment of the skin with zwitterionic EPC increases the iontophoretic transdermal mannitol flux about three-fold compared to iontophoretic control without pretreatment. However, skin pretreatment with another zwitterionic phospholipid, DSPC, did not influence the iontophoretic flux of mannitol. In contrast, pretreatment of the skin with cationic SA decreased the iontophoretic mannitol flow from the anode. It is concluded that EPC works as a penetration enhancer further increasing the transdermal mannitol flux during iontophoresis. In contrast, the cationic stearylamine changes the charge of the skin, thus leading to decreased electroosmosis and decreased mannitol flux. Hence, the effects of stearylamine are assumed to be mediated by the alterations in the charge of the stratum corneum structures, while EPC is suggested to decrease the permeability barrier of the skin.