The existence of an intracellular stage of Trypanosoma rangeli in the vertebrate host was evaluated by experimental infection of the U937 histiocytic cell line with the San Agustín strain and the Ub66-5b clone. The identity of the parasites at the beginning and end of the experiments was confirmed through biological behavior in the vector and mammal hosts, isoenzymes, polymerase chain reaction (PCR), and monoclonal antibodies. Infectivity to U937 cells of T. rangeli obtained from culture and salivary glands was evaluated under different experimental conditions. These included 34 C vs. 37 C, opsonized vs. nonopsonized parasites, and 2, 4, 6, 24, 48, and 72 hr of cell-parasite contact. Trypanosoma rangeli adopted a characteristic nondividing amastigote-like form within U937 cells, which was different in size (P = 0.001) from Trypanosoma cruzi amastigotes. Culture forms of T. rangeli were more infective than parasites from salivary glands (P = 0.049) but were less infective than T. cruzi (P = 0.0001). Variations in temperature (34-37 C) and complement opsonization did not affect infectivity. Viability of intracellular forms was confirmed by feeding Rhodnius prolixus with T. rangeli-infected cells. Resistance of T. rangeli to the intracellular milieu could be an important mechanism in producing chronic infections in mammals and in the infection of triatomines.