In this study, we analyzed the influence of proteinase expression on the cellular differentiation of Herpetomonas samuelpessoai. Along cellular differentiation, which was induced by dimethylsulfoxide (DMSO), the trypanosomatids secreted several molecules with variable proteolytic activity. All of them were inhibited by 10 mm 1,10-phenanthroline, suggesting that they are zinc-metalloproteinases. Analysis of parasite extracts revealed the occurrence of a 63-kDa metalloproteinase and a 45-kDa cysteine proteinase. After extraction with Triton X-114 followed by water-detergent partition, the 63-kDa component was present in both aqueous and detergent phases, which indicated that this enzyme may be distributed over different cellular compartments including membrane domains. The 45-kDa component, however, presented hydrophilic properties and was predominantly expressed by DMSO non-treated parasites, suggesting that proteinases may be involved in the process of cellular differentiation in H. samuelpessoai. This was confirmed by the fact that a cysteine proteinase inhibitor abrogated parasite differentiation. The role of proteinases and their relevance in the differentiation of H. samuelpessoai are discussed.