In vivo, epimastigotes of Trypanosoma cruzi colonize a lipidic superficial layer of the rectal cuticle of the vector Triatoma infestans. In vitro, epimastigotes of four cultured strains and one strain from reduviids use a terminal area of the flagellum to attach to a variety of artificial hydrophobic substances, such as hydrocarbons and a range of synthetic plastics. Trypomastigotes did not attach to these substrates. Hydrophilic molecules, such as neutral or negatively charged polysaccharides, did not facilitate binding. Epimastigotes and trypomastigotes were artificially bound by electrostatic forces to positively charged chitosan or DEAE-Sephacel over their entire surface. Tween 20 and lipid-binding serum albumin effectively inhibited the hydrophobic attachment. Based on this hydrophobic interaction of epimastigotes, a new chromatography technique has been devised to gently separate trypomastigotes from epimastigotes using octacosane-coated beads. Furthermore, the in vitro transformation of epimastigotes to trypomastigotes was enhanced if epimastigotes were permitted to attach to hydrophobic, wax-coated culture vessels.