Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-alpha, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed.