We have analyzed the total cell extract, cell surface, and secretory protein profiles related to cellular differentiation triggered by dimethylsulfoxide in the insect trypanosomatid Herpetomonas samuelpessoai. The flagellates were cultivated in chemically defined conditions in the absence or in the presence of 4% DMSO, and the resolved protein bands were detected by SDS-PAGE gels and avidin-Western blotting. The cell-associated proteins showed a complex pattern of around 40 silver staining bands ranging from 15 to 150 kDa. There were generally minor quantitative differences in the protein profile between the non-treated and the DMSO-treated cells. The cell-surface protein profile revealed by the incubation of live parasites with biotin showed a decrease in the expression of the 120 kDa biotinylated polypeptide observed in the DMSO-treated cells when compared with untreated ones. However, control samples of both systems showed an endogenous biotinylated polypeptide of 63 kDa which also presented gelatinolytic activity. The trypanosomatids released at least 10 polypeptides to the culture medium. A low molecular mass exopolypeptide (35 kDa) was found exclusively in untreated cells, whereas a high-molecular-mass exopolypetide (250 kDa) was preferentially found in DMSO-treated cells.