The goal of the present study was to evaluate the antileishmanial effects of topically applied lipid-based formulations containing amphotericin B (AmB) in CBA mice as a model for human cutaneous leishmaniasis. Such treatment, if efficacious, is expected to be superior to systemic treatments since, by acting in a localized manner, it will require lower, and therefore less toxic, drug dosages. Three preparations of AmB complexed to polar lipids were tested: Fungizone (mixed micelles composed of AmB and deoxycholate), Amphocil (AmB and cholesteryl sulfate complex), and ABPLC (AmB and phospholipid complex). All these formulations killed parasites in vitro with similar efficacies but were ineffective when they were applied topically. However, Amphocil and ABPLC, but not Fungizone, when dispersed in an aqueous solution containing 5 to 25% ethanol, induced a statistically significant improvement in lesion size from week 2 or 3 onward (a total of 15 mg of AmB per kg of body weight was applied over 3 weeks). AmB biodistribution measurements following topical application of Amphocil, determined by high-pressure liquid chromatography, showed that AmB was detectable in the skin but not in the internal organs. Application of at least 10 times more drug was necessary to obtain detectable levels of AmB in the internal organs. After application of therapeutic doses of ABPLC, very low levels of AmB were detected in the internal organs. These experiments show for the first time that AmB administered topically as a complex either with cholesteryl sulfate or with phospholipids and in the presence of ethanol can penetrate the skin and kill sensitive organisms in a localized manner by using very low total drug concentrations.