ABSTRACT Leishmania mexicana is a protozoan parasite that causes a disease in humans with frequent relapses after treatment. It is also highly resistant to the currently available drugs. For this reason, there is an urgent need for more effective antileishmanial drugs. Hydroxyurea, an anticancer drug, is toxic to replicating eukaryotic cells and has been proven to be effective in arresting the Leishmania major cell cycle. In this study, hydroxyurea was tested in an in vitro model of intracellular Leishmania infection in macrophages. The parasite density in infected macrophages was measured by microscopy after incubation for various times and treatment with hydroxyurea at different concentrations. Viable parasites that could be transformed into promastigotes by shifting the temperature to 26°C were counted every other day after the replacement of hydroxyurea with fresh medium. Meglumine antimoniate, the standard drug treatment for Leishmania mexicana , was used as a reference drug under the same experimental conditions. Hydroxyurea completely eliminated Leishmania parasites when it was used at a dosage of 10 or 100 μg/ml. Differences in the length of treatment needed to achieve elimination were as follows: the 10-μg/ml doses required 9 days, while 3 days was sufficient when 100 μg/ml was used. Hydroxyurea had a 50% effective dose of 0.015 μg/ml in vitro, which was observed on day 6 after exposure. Hydroxyurea is highly effective in killing intracellular amastigotes in vitro.