The members of the PUF family of RNA-binding proteins regulate the fate of mRNAs by binding to their 3'UTR sequence elements in eukaryotes. In trypanosomes, for which gene expression is polycistronic and controlled almost exclusively by post-transcriptional processes, PUF proteins could play a crucial role. We report here the complete analysis of the PUF protein family of Trypanosoma cruzi composed of 10 members. In silico analysis predicts the existence of at least three major groups within the T. cruzi family, based on their putative binding specificity. Using yeast three hybrid assays, we tested some of these predictions for TcPUF1, TcPUF3, TcPUF5, and TcPUF8 as representatives of these groups. Data mining of the T. cruzi genome led us to describe putative binding targets for the TcPUFs of the most conserved group, TcPUF1 and TcPUF2. The targets include genes for mitochondrial proteins and protein kinases. Finally, immunolocalization experiments showed that TcPUF1 is localized in multiple discrete foci in the cytoplasm supporting its proposed function.