Our confocal microscopy observations indicated a weaker, punctate R123 GSI-IX molecular weight signal along the mitochondrial membrane, suggesting that the H + electrochemical gradient was weaker in drug-treated parasites. The greater R123 fluorescence intensity in treated parasites on flow cytometry may therefore be due to the branching of the inner mitochondrial membrane, resulting in higher levels of R123 accumulation within cells. The observed circularization of mitochondrial cristae may result from the disorganization and subsequent fusion of inner membranes in the absence of mature endogenous sterols. In this context, the presence of myelin figures in close contact with mitochondria may indicate the degradation of damaged mitochondrial membranes by mitophagy and/or the involvement of mitochondrial membranes in autophagosome assembly, as recently demonstrated in starvation-induced autophagy. The possible involvement of mitochondria in T. cruzi autophagosome biogenesis requires further investigation. We are currently producing T. cruzi cell lines expressing fluorescent proteins tagged to the autophagosome marker ATG-8 as a tool for addressing this issue. By contrast, given the mitochondrial location of the mevalonate pathway enzymes HMG-CoA synthase and HMG-CoA reductase in T. cruzi and the presence of endogenous sterols in the inner membrane of this organelle, mitochondrial inner membrane branching in response to SBIs at the EC50/72 h may be a direct response to the depletion of endogenous sterols, highlighting the importance of this organelle in the ergosterol biosynthesis of trypanosomatids. After longer periods of drug exposure, higher levels of branching were observed with lovastatin than with ketoconazole, possibly because the target of lovastatin is located in the mitochondrion, whereas that of ketoconazole is located in the endoplasmic reticulum and reservosomes. Another morphological change observed in response to treatment with ketoconazole or lovastatin at the EC50/72 h was an increase in reservosome size. Reservosomes are found exclusively in the Schizotrypanum subgenus, in which they take the form of spherical organelles concentrated in the posterior PB 203580 region of T. cruzi epimastigotes; they are thought to be prelysosomal compartments in which material from endocytosis accumulates. No typical lysosomes have ever been found in T. cruzi, so the reservosomes were recently given the name ����lysosome-related organelles����, due to their acidic pH and the presence of acidic hydrolases. The progressive hypertrophy of this organelle in the face of sterol inhibition was recently demonstrated following treatment with ketoconazole. We show here that reservosome size also increases in response to lovastatin. A recent analysis of reservosome content by mass spectrometry showed the presence of two enzymes responsible for the final steps in ergosterol biosynthesis. Together with the recent demonstration of the presence of C14-DMT in this organelle, it seems that the increase in reservosome size following SBI treatment reflects the involvement of this organelle in sterol biosynthesis. As for mitochondrial branching, the more intense swelling of the reservosome observed after treatment with ketoconazole than after treatment with lovastatin may reflect differences in the cellular distributions of the targets of these drugs. However, the abnormal increase in reservosome size may also be due to the autolysomal function of this organelle, as the material engulfed by autophagsomes is delivered to this organelle for degradation during autophagy. In this context, reservosome hypertrophy and mitochondrial branching may be signals of intense autophagy.