ABSTRACT.    The Trypanosoma cruzi cysteine protease cruzain is the target of novel chemotherapy for Chagas' disease. The biological role for cruzain in immune evasion was elucidated in a comparative study of parental wild type-parasites and parasites resistant to the cysteine protease inhibitor N-Pip-F-hF-Vsφ that results in protease-deficiency. Wild type T. cruzi does not activate the host macrophage following infection. Cruzain and the signaling factor NF-κB P65 co-localize to the cell surface of intracellular parasites. P65 is proteolytically cleaved. No significant IL-12 expression occurs in macrophages infected with wild type parasites and subsequently activated with LPS confirming macrophage unresponsiveness. In contrast, cysteine protease inhibitor-resistant, and cruzain-deficient parasites induce macrophage activation and nuclear NF-κB P65 localization. Thus, cruzain hinders macrophage activation allowing T. cruzi survival and replication, and expansion of infection in Chagas' disease.