The ruthenium NO donors of the group trans-[Ru(NO)(NH3)4L]n+, where the ligand (L) is N-heterocyclic H2O, SO(3)(2-), or triethyl phosphite, are able to lyse Trypanosoma cruzi in vitro and in vivo. Using half-maximal (50%) inhibitory concentrations against bloodstream trypomastigotes (IC50try) and cytotoxicity data on mammalian V-79 cells (IC50V79), the in vitro therapeutic indices (TIs) (IC50V79/IC50try) for these compounds were calculated. Compounds that exhibited an in vitro TI of > or = 10 and trypanocidal activity against both epimastigotes and trypomastigotes with an IC50(try/epi) of < or = 100 microM were assayed in a mouse model for acute Chagas' disease, using two different routes (intraperitoneal and oral) for drug administration. A dose-effect relationship was observed, and from that, the ideal dose of 400 nmol/kg of body weight for both trans-[Ru(NO)(NH3)4isn](BF4)3 (isn, isonicotinamide) and trans-[Ru(NO)(NH3)4imN](BF4)3 (imN, imidazole) and median (50%) effective doses (ED50) of 86 and 190 nmol/kg, respectively, were then calculated. Since the 50% lethal doses (LD50) for both compounds are higher than 125 micromol/kg, the in vivo TIs (LD50/ED50) of the compounds are 1,453 for trans-[Ru(NO)(NH3)4isn](BF4)3 and 658 for trans-[Ru(NO)(NH3)4imN](BF4)3. Although these compounds exhibit a marked trypanocidal activity and are able to react with cysteine, they exhibit very low activity in T. cruzi-glycosomal glyceraldehyde-3-phosphate dehydrogenase tests, suggesting that this enzyme is not their target. The trans-[Ru(NO)(NH3)4isn](BF4)3 and trans-[Ru(NO)(NH3)4imN](BF4)3 compounds are able to eliminate amastigote nests in myocardium tissue at 400-nmol/kg doses and ensure the survival of all infected mice, thus opening a novel set of therapies to try against trypanosomatids.