Autoreactive T-cells can be activated inadvertently during immune responses through antigen-independent pathways. It has been suggested that Fas/Fas ligand interactions may play a role in eliminating these cells, but the extent that cells activated through such alternative pathways are sensitive to Fas-induced apoptosis has not been extensively evaluated. Proliferation of peripheral blood T-cells from normal individuals activated for 4 days with PHA or PMA + ionophore was not influenced by the presence of anti-Fas antibody. When the same cells were activated with soluble factors produced by previously activated T-cells (lymphostimulatory activity), anti-Fas antibodies inhibited thymidine incorporation by 74+/-4%. The presence of typical morphological changes and oligonucleosomal fragmentation of DNA indicated that the reduced proliferation resulted from apoptotic death of the lymphoblasts. Fas-sensitivity of T-cells activated by lymphostimulatory activity was first detectable 4 days after activation, and at 5 days the majority of lymphoblasts had become sensitive to Fas, whereas no evidence of sensitivity to Fas was observed for lymphoblasts generated by PHA or PMA + ionophore during the first 5 days of culture. Incubation of cells activated with PHA or PMA+ ionophore in the presence of IL-2 at concentrations 10-fold higher than that present in lymphostimulatory activity did not induce early sensitivity to Fas, indicating that exposure to IL-2 could not explain the precocious development of sensitivity to Fas seen following activation by lymphostimulatory activity. These studies demonstrate that T-cells activated through an antigen-independent 'alternative' pathway develop precocious sensitivity to Fas-induced apoptosis, which may be important in permitting the elimination of autoreactive bystander cells activated in the course of immune responses.