PURPOSE: Peptidyl-tRNA hydrolase (Pth), first described in Escherichia coli, is responsible for rescuing stalled ribosomes during peptidyl-tRNA "drop off". Bacterial Pth has been widely studied, but the characterization of eukaryotic Pth remains a poorly researched field, especially in protozoan parasites. This work aimed to characterize Trypanosoma cruzi Pths and determine their localization. METHODS: Two open reading frames (ORFs) that may encode Pths were identified in the T. cruzi genome. Bioinformatics analysis was performed for each protein using conserved domain analysis and multiple alignment. ORFs were cloned into an expression vector, E. coli pth(Ts) competent cells were transformed, and thermosensitivity tests were performed. Recombinant proteins were expressed and purified to immunize rats and obtain polyclonal antibodies. Pull down and immunoprecipitation followed by mass spectrometry to verify the interactions. RESULTS: TcPth and TcPth2 have a conserved domain corresponding to the Pth2 superfamily. Multiple alignments with previously characterized amino acid sequences of Pths showed that they are unrelated to T. cruzi proteins, considering that conserved residues of catalytic importance are absent. TcPth was able to rescue the E. coli thermosensitive pth(Ts) mutation, but TcPth2 was not. TcPth2 interacts with reservosome proteins such as cysteine peptidase and endocytic pathway proteins. CONCLUSION: The results suggest that TcPth and TcPth2 has a different function. This work represents the first in its area since the Pths of the T. cruzi were characterized and breaks ground for the characterization of Pths from other protozoan parasites.