Improved rationally designed lead drug structures against African trypanosomiasis, Chagas disease, and leishmaniasis were obtained against trypanothione reductase from Trypanosoma cruzi. Substituted-benzyl [3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)propyl]dimethylammonium salts, synthesized by Menschutkin quaternization of the tertiary alkylamine ω-nitrogen atom of chlorpromazine, were linear, competitive inhibitors of recombinant trypanothione reductase from T. cruzi, with either trypanothione disulfide or N-benzyloxycarbonyl-Lcysteinylglycyl 3-dimethylaminopropylamide disulfide as substrate. The permanent positive charge on the distal nitrogen atom of the tricyclic side chain contribution to binding was estimated as g5.6 kcal‚mol-1 by comparison with the analogue with the cationic nitrogen atom of the quaternary replaced by an ether oxygen atom. A further major contribution to improving Ki values and inhibition strength was the hydrophobic natures and structures of the N-benzyl substituents. The strongest inhibitor, the [3-(2-chloro-4a,10a-dihydrophenothiazin-10-yl)propyl]- (3,4-dichlorobenzyl)dimethylammonium derivative (Ki 0.12 µM), was ∼2 orders of magnitude more inhibitory than the parent chlorpromazine. Several of these quaternary phenothiazines completely inhibited T. brucei parasite growth in vitro at < 1 µM). Although active against Leishmania donovani, none of the analogues showed major improvement in this activity relative to chlorpromazine or other nonquaternized phenothiazines. The p-tert-butylbenzyl-quaternized analogue very strongly inhibited (ED50 < 1 µM) growth of the amastigote stage of T. cruzi.