Incubation of mouse thymocytes with adenosine and its receptor site agonist, 2-chloroadenosine, induced a pronounced increase in the intracellular cAMP level and resulted in internucleosomal DNA fragmentation followed by cell lysis. Similar DNA fragmentation was induced in peripheral T-lymphocytes prepared from spleen cells but to a lesser extent than in the thymocytes. The DNA fragmentation in both thymocytes and splenic T-lymphocytes was prevented by the addition of actinomycin D and cycloheximide, indicating that this process required mRNA and protein synthesis. The inhibition was accompanied by a reduction in cell lysis as judged by the release of lactate dehydrogenase into the medium. Involvement of cAMP accumulation in inducing DNA fragmentation was supported by the results of experiments with cAMP analogs such as dibutyryl cAMP and 8-bromo-cAMP, and cAMP level-raising drugs including forskolin, cholera toxin, and isobutylmethyxanthine. The latter agents induced pronounced or sustained elevation of cellular cAMP followed by internucleosomal DNA cleavage in T-lymphocytes. These results suggest that adenosine receptor-mediated accumulation of cyclic AMP regulates T-lymphocyte death through inducement of internucleosomal DNA cleavage.