This study provides several pieces of evidence indicating that 3F6-Ag, identified by monoclonal antibody (MAb) 3F6 as a stage-specific glycoprotein of approximately 82 kDa on the surface of metacyclic trypomastigotes of different Trypanosoma cruzi strains, promotes the entry of parasites into host cells through a ligand-receptor type interaction. First, invasion of Vero cells by metacyclic trypomastigotes of both CL and Tulahuen strains was significantly inhibited by MAb 3F6 or its Fab fragments. Second, purified 3F6-Ag bound to Vero cells in a dose-dependent and saturable fashion. Third, soluble 3F6-Ag reduced the infection of Vero cells by metacyclic forms of CL and Tulahuen strains by 90 to 97 and 50%, respectively. Unrelated proteins, as well as extracellular matrix components, such as heparan sulfate and collagen, had no effect. Our studies also show that in the Tulahuen strain, 10D8-Ag, a 35/50-kDa glycoprotein identified by MAb 10D8, participates in target cell invasion, confirming previous observations, but the variant form of 10D8-Ag expressed by highly invasive CL strain metacyclic trypomastigotes appears to be irrelevant. Overall, our results indicate that the surface components of T. cruzi metacyclic trypomastigotes involved in the process of host cell penetration are developmentally regulated molecules, such as 3F6-Ag and 10D8-Ag, that have no counterpart in blood- or tissue culture-derived trypomastigotes.