The most potent trypanocidal compound of a series of symmetrically substituted 1,4-bis(3-aminopropylpiperazines) which displayed an IC50 value of 5 microM on Trypanosoma cruzi trypomastigotes, was inactive on trypanothione reductase. Two derivatives 6 and 12 of this compound, one symmetrical and one dissymmetrical, were synthesized via a reductive amination reaction, to prepare affinity chromatography columns, which allowed us to isolate three parasitic proteins. Among these, the major ligand 6- and 12-binding protein having an apparent molecular weight of 52 kDa has been identified as the thiol-disulfide oxido-reductase Tc52, previously characterized in Trypanosoma cruzi.