A number of bis(benzyl)polyamine analogs were found to be potent inhibitors of Leishmania donovani growth in vitro (IC50 = 4.3-25 microM). Structural variations appear to have important effects on the biological functions of these analogs. Subinhibitory concentration of all of the analogs with the exception of MDL 27994 could rescue the cells from DL-alpha-difluoromethylornithine toxicity. The analogs inhibited macromolecular synthesis as evaluated by [3H]thymidine, [14C]uracil, and [35S]methionine incorporation. They inhibited the activity of the two enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), involved in the biosynthesis of polyamines. These analogs depleted intracellular levels of natural polyamines. We conclude, therefore, that the major mechanism by which these analogs act may be by disruption of macromolecular biosynthesis and cell death. Repression of polyamines by these analogs may be yet another factor involved in slowing the growth of the parasite.