Trypanosoma cruzi colonizes mainly the rectum of the vector, especially the rectal glands. We investigated the basic architecture of the rectal cuticle of Triatoma infestans and the mode of attachment of T. cruzi in the small intestine and the rectum. In addition, we determined the capacity of culture-derived epimastigotes to attach to artificial substrates and the influence of attachment on metacyclogenesis. After incubation of the rectum with wheat germ lectin (WGA) coupled to gold particles, the procuticle contained chitin, but the two layers of the epicuticle and the superficial layer bordering the rectal lumen did not. The specific fluorochrom Nile Red stained the entire rectal cuticle green, indicating the waxy composition of the superficial layer. In electron microscopic analysis the parasites were attached to the hydrophobic superficial wax layer but not to the epithelium of the midgut. In vitro culture-derived epimastigotes attached with a high affinity to all hydrophobic substrates tested, whereas hydrophilic substrates did not permit attachment. Emulsified hexadecane localized the attachment molecules to the terminal part of the flagellum. Inhibition of attachment by coating the culture tubes with hydrophilic agarose and constant agitation decreased the rate of epimastigote to trypomastigote transformation, whereas wax coating enhanced metacyclogenesis.