Abstract Incubation for 24 h in culture medium containing 50mM adenosine triphosphate (ATP) produces distinct alterations in the ultrastructure of Trypanosoma cruzi epimastigotes, most obvious of which is the formation of large membrane-bound vacuoles in the cytosol. These vacuoles become positive following exposure to the macromolecule horseradish peroxidase (HRP). After a 20-min chase in phosphate-buffered saline (PBS) the HRP-positive vacuoles begin to separate into discrete structures such that after a 60-min chase, obvious reservosomes are identifiable. It is hypothesized that extracellular ATP causes increased permeability of the epimastigotes plasma membrane, resulting in ionic fluxes that, in turn, interfere with the normal formation of reservosomes.