Microtubule cytoskeleton is a dynamic structure involved in the maintenance of eukaryote cell shape, motion of cilia and flagellum, and intracellular movement of vesicles and organelles. Many antibodies against tubulins have been described, most of them against the C-terminal portion, which is exposed at the outside of the microtubules. By generating a novel set of monoclonal antibodies against the cytoskeleton of Trypanosoma cruzi, a flagellate protozoan that causes Chagas' disease, we selected a clone (mAb 3G4) that recognizes β-tubulin. The epitope for mAb 3G4 was mapped by pepscan to a highly conserved sequence motif found between α-helices 11 and 12 of the C-terminus of β-tubulin in eukaryotes. It labels vesicular structures in both T. cruzi and mammalian cells, colocalizing respectively with a major cysteine protease (Cruzipain) and lysosome associated protein (LAMP2) respectively, but it does not label regular microtubules on these cellular models. We propose that the epitope recognized by mAb 3G4 is exposed only in a form of tubulin associated with endosomes.