The surface anionic groups of untreated or dimethyl sulfoxide (DMSO)-treated Herpetomonas samuelpessoai cells were analyzed by cell electrophoresis, ultrastructural cytochemistry, and identification of sialic acids using thin-layer chromatography. Differentiation of H. samuelpessoai induced by DMSO treatment caused a significant increase in the net negative surface charge. In flagellates exposed to DMSO, more cationized ferritin, colloidal iron hydroxide, and sendai virus particles bound to the cell surface. Treatment of both untreated and DMSO-treated flagellates with neuraminidase decreased markedly the EPM of cells to the cathodic pole. These findings suggest that sialic acid residues are the major anionogenic groups exposed on the surface of H. samuelpessoai. Thin-layer chromatography showed that N-acetyl and N,O-diacylneuraminic acids, in equal proportions, were present in H. samuelpessoai. However, N-acetylneuraminic acid predominates in DMSO-treated cells.