The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding beta-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi beta-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.