Stimulation via the CD3/TCR molecular complex induces proliferation of resting T cells, but triggers programmed cell death (apoptosis) in immature thymocytes and preactivated mature T cells. Activation-induced cell death (AICD) triggered by anti-CD3/TCR mAb or by staphylococcus enterotoxin superantigen is associated with fragmentation of genomic DNA into oligonucleosomal fragments of 200 bp length, thus displaying the characteristic features of apoptosis. Here, we show that a fraction (20-50%) of cells in alloreactive CD8 human short-term T cell lines, generated by repeated restimulation with EBV-transformed B cell lines, undergo AICD when restimulated with the appropriate (but not with third party) stimulator cells. AICD of responder T cells is inhibited when stimulator cells are preincubated with anti-HLA class I mAb but not with anti-HLA class II mAb, indicating that T cell death is dependent on alloantigen (HLA class I) recognition by responding CD8 T cells. Importantly, alloantigen-induced T cell death occurs in the absence of detectable DNA fragmentation. Thus, several independent assay systems all failed to reveal low molecular weight DNA fragmentation, even though DNA fragmentation was readily detected in T cell lines exposed to PHA or gamma-irradiation. Alloantigen-induced T cell death was prevented by aurintricarboxylic acid, which has previously been shown to inhibit apoptosis in experimental systems where no DNA fragmentation occurs. Taken together, these results demonstrate that alloantigen can trigger AICD in mature responding T cells in the absence of low molecular weight DNA fragmentation.