A variety of culture systems have been developed to study mechanisms of activation-induced cell death in peripheral T lymphocytes either during the initial period after exposure to an activating stimulus or following repeated stimulation of activated T cells. In this study we describe a new culture model for the analysis of apoptosis after withdrawal of TCR signals from activated T cells. T cells activated by anti-CD3 antibodies for 48 h and then further cultured in the presence of IL-2 but absence of continued CD3/TCR stimulation underwent dramatic cell death approximately 4 days following removal of the TCR stimulus. Apoptotic cells generated in this protocol, unlike those produced by hyperstimulation, retained substantial levels of degraded DNA following fixation, consistent with death in the G0/G1 phase of the cell cycle. This "agonist withdrawal" cell death occurred largely within the CD8 T cell subset, with CD4 cells showing lower levels of apoptosis. This form of cell death did not appear to be the result of IL-2 exhaustion, since repeated addition of IL-2 during the culture period did not significantly alter the number of apoptotic cells. Apoptosis induced by agonist withdrawal was not blocked by Fas antigen fusion protein or by anti-TNF alpha-neutralizing antibodies, suggesting a mechanism independent of Fas/FasL and TNF alpha/TNF-R interactions. Cell death was, however, significantly inhibited by treatment with a CD30 fusion protein. CD30 was found to be transiently expressed on CD8 T cells immediately prior to death, with lower expression on CD4 cells, while CD30 ligand was found to be expressed most strongly by CD4 T cells. These results suggest a role for CD30 in regulating the onset of apoptosis in CD8 T cells after interruption of CD3/TCR.