With the aim of investigating if delivery of benznidazole (BNZ) to liver could be increased by incorporating the drug in multilamellar liposomes, single bolus of free BNZ or liposomal BNZ formulations (MLV-BNZ) composed of HSPC:DSPG:Chol 2:1:2 (mol/mol/mol) at 0.7% (w/w) drug/total lipid ratio, were injected by intramuscular (i.m.), subcutaneous (s.c.) and intravenous (i.v.) routes, at 0.2 mg BNZ/kg, in rats. The resulting blood concentrations were followed along 9 h post-injection (p.i.) and drug accumulation in liver was determined after 4 and 9 h p.i. Only upon i.v. injection of MLV-BNZ, a threefold higher BNZ accumulation in liver was obtained, together with blood BNZ concentrations of 1.1 μg/ml (30% lower than the blood BNZ concentration achieved upon i.v. administration of free drug) occurred 4 h p.i. However, such increased liver uptake of BNZ, raised twice a week had no effect on parasitaemia levels of mice infected with the RA strain of Trypanosoma cruzi. Our results indicate that the relationship between increased selectivity for an infected tissue and therapeutic effect is not always straightforward, at least for the MLV-BNZ regimen used in the present study.