We analysed the proteinase profiles in nine Trypanosoma (Herpetosoma) rangeli strains from distinct geographical regions, three T. (Schizotrypanum) cruzi reference strains and in T. (Herpetosoma) lewisi, T. (Herpetosoma) musculi and T. (Megatrypanum) conorhini samples by detection of enzyme activity on SDS-PAGE containing co-polymerized gelatin as substrate. Three different profiles of cysteine proteinase activity were detected in T. rangeli within a similar range of molecular mass. T. lewisi and T. musculi displayed a similar pattern of proteolysis, quite distinct from the ones detected in T. rangeli and in the other subgenera belonging to the stercorarian trypanosomes. Our results indicate the potential of the SDS-PAGE-gelatin technique in distinguishing between these parasites, and confirm several findings on the existence of subgroups within the T. rangeli taxon. These may represent either subspecies or distinct species. Our results also demonstrate the lack of affinity of T. rangeli with the species belonging to the subgenus Herpetosoma.