Trypanosoma cruziis the ethiological agent of Chagas disease. New compounds are being developed based on the biosynthesis and function of sterols, becauseT. cruzihas a requirement for specific endogenous sterols for growth and survival. Sterol biosynthesis inhibitors (SBIs) are drugs commonly used against fungal diseases. These drugs act by depleting essential and specific membrane components and/or inducing the accumulation of toxic intermediary or lateral products of the biosynthetic pathway. In this work we present the effects of WSP488, WSP501, and WSP561, specific inhibitors of Δ²⁴⁽²⁵⁾-sterol methyl transferase, on the ultrastructure ofT. cruziepimastigotes. All three drugs inhibited parasite multiplication at low concentrations, with IC₅₀values of 0.48, 0.44, and 0.48 μM, respectively, and induced marked morphological changes including (a) blockage of cell division; (b) swelling of the mitochondrion, with several projections and depressions; (c) swelling of the perinuclear space; (d) presence of autophagosomes and myelin-like figures; (e) enlargement of the flagellar pocket and of a cytoplasmic vacuole located in close association with the flagellar pocket; (f) detachment of the membrane of the cell body; and (g) formation of a vesicle at the surface of the parasite between the flagellar pocket and the cytostome. Our results show that these drugs are potentin vitroinhibitors of growth ofT. cruzi.