The epimastigotes (insect stage) of Trypanosoma cruzi, are unable to synthesize de novo the diamines putrescine and its analogue cadaverine, from their amino acid precursors. Therefore the metabolic pathways to polyamines (aliphatic bases) in T.cruzi, clone XI0/6 epimastigotes, have been studied by in vitro radiolabelling using these diamine precursors. [3H]Putrescine was rapidly taken up from the medium and incorporated in to the polyamines spermidine, spermine and the glutathione-polyamine conjugate N1, N2-bis(glutathionyl)spermidine(trypanothione). Likewise [3H]cadaverine was rapidly taken up by T.cruzi and converted into the analogous polyamines aminopropylcadaverine and bis(aminopropyl) cadaverine and the glutathione-polyamine conjugates glutathionylaminopropylcadaverine and N1, N2-bis(glutathionyl)- aminopropylcadaverine (homotrypanothione). Detailed analysis has revealed that T.cruzi epimastigotes (clone X10/6) transport exogenous [3H]putrescine and [3H]cadaverine by a rapid, high affinity, temperature dependent, diamine transport system which exhibits saturable kinetics (putrescine Km, - 2.0 μM, Vmax- 3.3nmol min-1 (10(8) cells)-1; cadaverine Km= 13.4 μM, V ■ 3.9 nmol min-1 (10(8) cells)-1). Diamine transport requires the presence of a proton gradient and thiol groups, does not utilise an amino acid transporter and its activity is altered as die cells proceed through the growth cycle. This transporter shows high specificity for the diamines, putrescine and cadaverine, but low specificity for the polyamines, spermidine and spermine. Hence polyamine metabolism in T.cruzi epimastigotes differs from other trypanosomadids ('Trypanosoma brucei, Leishmania and Crithidia fascicuiata) in three ways. Firstly T.cruzi lacks the ability to synthesize diamines de novo. Secondly both putrescine and cadaverine are rapidly taken up and can be converted into significant amounts of spermine and bis(aminopropyl) cadaverine respectively. Thirdly T.cruzi is able to synthesize homotrypanothione in addition to trypanothione. If the pattern of polyamine metabolism in the mammalian stages of T.cruzi is similar to that observed with the epimastigotes, these findings will have important implications with respect to future developmental strategies for the chemotherapy of Chagas’ disease.