Trypanosoma cruzi trypomastigotes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) in vitro are rapidly induced to differentiate into round forms. Using confocal microscopy, we were able to show that trypomastigotes treated with PI-PLC initiate the process of flagellum remodeling by 30 sec after contact with the enzyme and amastigote-like forms are detected as early as 10 min after PI-PLC treatment. Scanning and transmission electron microscopy indicate that trypomastigotes undergo a previously undescribed process of flagellum circularization and internalization. Analysis of the flagellar complex with monoclonal antibody 4D9 shows heterogeneous labeling among the parasites, suggesting a remodeling of these molecules. After PI-PLC treatment, parasites rapidly lose the surface marker Ssp-3 and 24 h post-treatment they begin to exhibit a circular nucleus and a rod-shaped kinetoplast. By flow cytometry analysis and confocal microscopy, the Ssp-4 amastigote-specific epitope can be detected on the parasite surface. This indicates that the release of trypomastigote GPI-anchored molecules by exogenous PI-PLC in vitro can trigger morphological changes.