Background: Iontophoresis can enhance penetration of drugs into tissues. We examined the extent of penetration of gentamicin into the cornea of rats during iontophoresis and the effect of varying the concentrations of gentamicin, the duration of iontophoresis and the current densities during iontophoresis. Methods: Eight groups of rats underwent corneal iontophoresis using gentamicin dissolved in agar. Low and high concentrations of gentamicin were used, as well as low and high current densities and long and short durations of iontophoresis. Control groups received topical or subconjunctival gentamicin, topical saline solution and mock iontophoresis with the agar-gentamicin mixture. The Mann-Whitney test was used for statistical evaluation. Results: Highly bactericidal concentrations of gentamicin were obtained in all the iontophoresis-treated corneas. The high concentration compared to the low concentration of gentamicin in agar significantly increased the concentration of gentamicin in the corneas, as did the longer duration of iontophoresis. However, higher current intensity did not significantly enhance the drug concentration in the cornea. Conclusion: lontophoresis with a concentrated gentamicin-agar mixture may provide a rapid increase of levels in the cornea.