Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities, and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. To determine the possible role that proteasomes may play in controlling the life cycle of African trypanosomes, we have isolated proteasomes from the bloodstream and the insect (procyclic) forms of Trypanosoma brucei by DEAE-cellulose chromatography and glycerol gradient fractionation in the presence of ATP. No 26 S proteasome homologs was identified in T. brucei under these experimental conditions. The proteasomes isolated from these two forms of T. brucei are very similar to the rat blood cell 20 S proteasome in their general appearance under the electron microscope. The profile of trypanosome proteasome subunits in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has eight visible protein bands with molecular weights ranging from 23 to 34 kDa, and cross-reacted very poorly with the anti-human 20 S proteasome antibodies on immunoblots. Two-dimensional gel electrophoresis of the parasite proteasomes shows a similar number of major subunits with pI's ranging from 4.5 to 7. Using a variety of fluorogenic peptides as substrates, the trypanosome proteasomes exhibited unusually high trypsin-like, but somewhat lower chymotrypsin-like activities, as compared to the rat 20 S proteasome. These proteolytic activities were, however, insensitive to phenylmethylsulfonyl fluoride (PMSF), tosyl-phenylalanine chloromethylketone (TPCK), tosyl-lysine chloromethylketone (TLCK) and trans-epoxy succinyl-L-leucylamido-(4 guanidino) butane (E-64), but the trypsin-like activity of trypanosome proteasomes was inhibited by leupeptin, an aldehyde known to inhibit the trypsin-like activity of mammalian proteasomes, thus ruling out possible contamination by other serine or cysteine proteases. Some quantitative differences in the substrate specificities between the proteasomes from bloodstream and procyclic forms were indicated, which may play a role in determining the differential protein turnovers at two different stages of development of T. brucei.