SUMMARYIn a screening of 65 derivatives of natural quinones using bloodstream trypomastigotes of Trypanosoma cruzi, the 3 naphthoimidazoles derived from β-lapachone – N1, N2 and N3 – were selected as the most active. Investigation of their mode of action led to the characterization of mitochondrion, reservosomes and DNA as their main targets, and stimulated further studies on death pathways. Ultrastructural analysis revealed both autophagic (autophagosomes) and apoptotic-like (membrane blebbing) phenotypes. Flow cytometry analysis showed, in N2-treated trypomastigotes, a small increase of phosphatidylserine exposure, and a large increase in the percentage of necrosis, caused by N1 or N2. These death phenotypes were not detected in treated epimastigotes. The strong increase in labelling of monodansyl cadaverine, the inhibition of the death process by wortmannin or 3-methyladenine, the overexpression of ATG genes in treated epimastigotes, together with ultrastructural evidence point to autophagy as the predominant phenotype induced by the naphthoimidazoles. However, there are other pathways occurring concomitantly with variable intensities, justifying the need to detail the molecular features involved.