Proteasomes are intracellular complexes that control protein degradation in organisms ranging from Archaebacteria to mammals. In some parasitic protozoa, the proteasome is involved in cell differentiation and replication. In this study, we have used proteasome inhibitors to determine the biological role of proteasomes during the replication and in vitro metacyclogenesis of Trypanosoma cruzi. We used light and transmission electron microscopy to analyze morphological data and flow cytometry to analyze changes in the cell cycle. The growth of T. cruzi epimastigote culture forms in liver infusion tryptose medium was inhibited by the presence of up to 10 microM lactacystin. Inhibition was dose-dependent, with IC50 (50% inhibitory concentration) of 4.35 microM after 24 or 72 h. The metacyclogenesis process in vitro was strongly (95%) inhibited by 5 microM lactacystin treatment. The adhesion phase was not affected, but the epimastigotes did not differentiate into metacyclic trypomastigotes. Most treated epimastigotes had replicated DNA, with swelling of the mitochondrion and an altered distribution of nuclear and kinetoplast DNA. Our findings suggest that inhibition of the ubiquitin-proteasome pathway in T. cruzi epimastigotes does not block adhesion, but disrupts cell division and affects factors triggering differentiation.