Eupomatenoid-5, a compound isolated from leaves of Piper regnellii var. pallescens, showed antiprotozoal activity against the epimastigote proliferative stages and intracellular amastigote forms of Trypanosoma cruzi Y strain. Eupomatenoid-5, at 7.0 microg/ml (50% growth inhibition concentration) produced morphological changes in epimastigote forms of the parasite, such as intense cytoplasmic vacuolization, mitochondrial swelling, kinetoplast alteration, presence of myelin-like figures, and mitochondrial damage, as observed by transmission electron microscopy. In amastigote forms in LLCMK(2) cells, at 7.0-microg/ml concentration, the compound induced a decrease in the number of cells with internalized parasites and in the number of internalized parasites per LLCMK(2) cell, compared with untreated cells. Furthermore, intense cytoplasmic vacuolization and autophagic vacuoles were observed in T. cruzi intracellular amastigotes after 72 h of incubation. Scanning electron microscopy confirmed the alterations in the shape of the parasites.