Trypanothione reductase (TR) occurs exclusively in trypanosomes and leishmania, which are the etiological agents of many diseases. TR plays a vital role in the antioxidant defenses of these parasites and inhibitors of TR have potential as antitrypanosomal agents. We describe the syntheses of several spermine and spermidine derivatives and the inhibiting effects of these compounds on T. cruzi TR. All of the inhibiting compounds displayed competitive inhibition of TR-mediated reduction of trypanothione disulfide. The three most effective compounds studied were N4,N8-bis(3-phenylpropyl)spermine (12), N4,N8-bis(2-naphthylmethyl)spermine (14), and N1,N8-bis(2-naphthylmethyl)spermidine (21), with Ki values of 3.5, 5.5 and 9.5 microM, respectively. Compounds 12, 14, and 21 were found to be potent trypanocides in vitro with IC50 values ranging from 0.19 to 0.83 microM against four T. brucei ssp. strains. However, these compounds did not prolong the lives of mice infected with trypanosomes. This work indicates that certain polyamine derivatives which target a unique pathway in Trypanosomatidae have potential as antitrypanosomal agents.