Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles. Here, we use the recently completed genome sequences of T. cruzi (TcruziDB http://TcruziDB.org) in an in silico analysis to explore genes of proteasome-ubiquitin pathway in this parasite. We identified two clusters homologues to b5 gene that encoding the chymotrypsin-like subunit of 20S in TcruziDB. Its contains 936bp and encodes 311 amino acid residues with a calculated molecular mass of 34.2kDa. The predicted amino acid sequence of the trypanosomatid b5 shares respectively 83 and 70% overall identities with Trypanosoma brucei and Leishmania major. After that, in order to perform a comparative study of expression profile of the b5 gene, we used semiquantitative RT-PCR and epimastigotes from T. cruzi I (Colombian strain) and T. cruzi II (CL, CL-Brenner, Berenice-62, Berenice-78 and Y). Our results show similar levels of expression of b5 gene in all of the strains. After that, we studied the proteasome proteolytic activities associated to chymotrypsin-like by using the fluorogenic substrates SLLVT-MCA and an enriched fraction of the proteasome obtained by ultracentrifugation. Our results from this study demonstrated substantial differences in chymotrypsin-like activity between T.cruzi I and II strains, suggesting that post-translation modification which modulates the proteolytic activity in T. cruzi could be more efficient than transcriptional regulation. These hypotheses are now being investigated.