We describe here biochemical characterization of the 20S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of 7 - and 7 -subunits. Of the 7 -type subunits, 5 contain pro-sequences that are proteolytically removed during assembly and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three -subunits were labeled by the affinity reagents, only two -subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to 2 and 5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20S proteasome confirmed the nominal 1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11S regulator (PA26)-20S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.