Previous studies have shown that proteasome inhibitors are novel agents for chemotherapy of human African trypanosomiasis or sleeping sickness. In this study, five peptide trileucine methyl vinyl sulfones with different N-terminal substituents were tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. Two inhibitors displayed promising anti-trypanosomal activities with ED50 values in the sub-micromolar range. Higher trypanocidal activity of the compounds generally corresponded to a higher k(obs)/[I] value for inhibition of the trypsin-like activity but not for the inhibition of the chymotrypsin-like activity of the proteasome. These data suggest that inhibitors with strong activity against the trypsin-like activity of the proteasome are the rational choice for future anti-sleeping sickness drug development.