We have identified and purified a 74 kDa surface glycoprotein from heart myoblasts that specifically binds to Trypanosoma cruzi trypomastigotes, and inhibits the attachment and internalization of trypomastigotes into these cells. The native form of the 74 kDa glycoprotein was purified to apparent homogeneity by preparative scale isoelectrofocusing and anion exchange chromatography. Pre-incubation of trypomastigotes with soluble 74 kDa glycoprotein strongly inhibited the binding and internalization of trypomastigotes into heart myoblast monolayers in a concentration dependent-manner. Pre-incubation of heart myoblast monolayers with antibodies specific to the purified 74 kDa glycoprotein also strongly inhibited trypomastigote binding and internalization into heart cells in a concentration dependent manner. These results support the notion that the surface 74 kDa glycoprotein is a target molecule on heart myoblast cells to which T. cruzi binds in order to invade them.