Trypanosoma cruzi requires an intracellular environment to multiply within its mammalian host. We describe the purification and some properties of a protein secreted exclusively by the metacyclic (infective) forms of the parasite. This permeabilizing protein (relative molecular mass 64,000) was secreted under our experimental conditions only when the parasites interacted with HeLa cells, HeLa membranes, or wheat-germ lectin. The protein is thermostable, and its biological activity is inhibited by formaldehyde but not by ethanol or acetone. At low concentrations and over short treatment times, this protein acts as a permeabilizer and induces endocytosis. No significant protease or neuraminidase activity was found. When adsorbed onto bentonite particles and incubated in the presence of non-phagocytic cells the protein facilitated the penetration of the particles into the cells. Immune serum directed against the protein neutralized its cytotoxic action and reduced the rate of penetration of metacyclic forms into both macrophages and non-phagocytic cells. Our results suggest that the protein secreted by the parasite plays a key role in the penetration of its infective form into the host cell.