The purpose of the present work was to study the in vitro distribution of acyclovir in human skin layers after iontophoresis, applied in order to increase the amount of drug in the basal epidermis, site of Herpes simplex infections. Experiments were done with Franz diffusion cells applying, as donor, acyclovir solutions (pH values: 3.0 and 7.4) or a commercial cream. Quantification of drug at different skin depths was performed by horizontal slicing of frozen skin, and drug extraction and analysis by high-performance liquid chromatography. Seven h of transdermal iontophoresis (0.5 mA cm-2 induced an accumulation of acyclovir in epidermis and dermis ranging from 80 to 150 micrograms cm-3, characterized by homogeneous distribution of the drug in skin layers. After short current application time (30 min) however, the concentration profile of drug in skin was not significantly different from the obtained after seven h of passive diffusion, employing pH 3.0 donor solution. After 30 min of iontophoresis, the acyclovir reservoir on the skin was maintained for up to five h producing a dramatic increase of drug concentration in skin, evening out over 80 micrograms cm-3 until a depth of 300 micrograms. Acyclovir can be accumulated at target site more quickly and maintained at higher level through application of a iontophoretic pulse and by keeping the drug reservoir on skin.