The macrolide antibiotic megalomicin (MGM) has been shown to inhibit vesicular transport between the medial- and trans-Golgi, resulting in the undersialylation of cellular proteins (P. Bonay, S. Munro, M. Fresno, and B. Alarcón, J. Biol. Chem. 271:3719-3726, 1996). Due to the effects of MGM on the Golgi and on the replication of enveloped viruses, we decided to test whether it has any antiparasitic activity. The results showed that MGM has potent activity against the epimastigote stage of Trypanosoma cruzi, producing a 50% inhibitory concentration (IC₅₀) of 0.2 µg/ml. Furthermore, MGM was also active against the intracellular replicative, amastigote form of T. cruzi, completely preventing its replication in infected murine LLC/MK2 macrophages at a dose of 5 µg/ml. Although less potent, MGM was also active against Trypanosoma brucei epimastigotes (IC₅₀, 2 µg/ml) and Leishmania donovani and Leishmania major promastigotes (IC₅₀, 3 and 8 µg/ml, respectively). MGM also blocked intracellular replication of the asexual stage of Plasmodium falciparum-infected erythrocytes at 1 µg/ml. Finally, MGM was active in an in vivo model, resulting in the complete protection of BALB/c mice from death caused by acute T. brucei infection and significantly reducing the parasitemia. These results suggest that MGM is a potential drug for the treatment of veterinary and human parasitic diseases.