The pore-forming protein perforin is one of the main effector molecules which cytotoxic lymphocytes utilize to kill their targets both in vivo and in vitro. Natural killer cells and cytotoxic T lymphocytes play an important role in host defense against a number of intracellular microorganisms such as virus and protozoan, but the exact way they help control infection is unknown. On the other hand, many microorganisms have evolved successful escape strategies to avoid immune-cell-mediated attack. It is thus necessary to investigate the direct interaction of infectious microorganisms with the lytic machinery of cytotoxic lymphocytes and other cells. In the present work we report the effect of perforin on both a protozoan, Trypanosoma cruzi, and the infected host cell. Epimastigote, amastigote, and trypomastigote forms of T. cruzi, as well as infected macrophages, were assayed for their susceptibility to perforin based on three different criteria. T. cruzi in all three differentiation stages were resistant to purified perforin at doses up to 100-fold larger than that sufficient to kill susceptible tumor cells. No morphological change was observed under electron microscopy. Survival rates and infectivities of the treated parasites in vitro were similar to those of control parasites. Moreover, the measurement of calcium influx using Fura-2 to assess membrane damage revealed that T. cruzi resist perforin attack by avoiding transmembrane pore formation. Resistance to perforin was not transferred to host cells since infected macrophages could be easily destroyed by perforin while intracellular amastigotes remained intact.