Studies on the anti-trypanosomal factor (ATF) from Pseudomonas fluorescens revealed that the ultrastructure of Trypanosoma cruzi trypomastigotes was extensively damaged following treatment with the active fractions. Alterations were detected as early as 10 minutes after the initiation of treatment and were progressive as shown by the almost total destruction of the parasites after 10 hours. In vitro studies with cultured fibroblasts disclosed a slower penetration of the cultures by trypomastigotes which had been treated with the ATF. This effect was attributed largely to the decreased motility of the parasites produced by the ATF. Further studies on the chemical characterization of the ATF suggested that the active factor is structurally a hydrophobic acidic substance, possibly a small peptide with a blocked amino terminus.