The effects of the inhibitors of topoisomerase I and II, camptothecin and etoposide, as well as novobiocin and adriamycin, on the DNA fragmentation and viability of mouse thymocytes in primary culture were examined. All inhibitors were shown to produce dose-dependent internucleosomal DNA cleavage by resolving isolated DNA by agarose-gel electrophoresis. The DNA fragmentation seemed to precede cell death, determined on the basis of LDH release, by a few hours. Etoposide-induced DNA fragmentation progressively increased after incubation and was enhanced by pretreatment with phorbol 12,13-dibutyrate, a phorbol ester capable of activating protein kinase C, whereas camptothecin-induced DNA fragmentation increased progressively after 12 h incubation and was unaffected by phorbol 12,13-dibutyrate-pretreatment. The process was also energy-dependent and required RNA and protein synthesis and protein phosphorylation, since it was inhibited by sodium azide, actinomycin D, cycloheximide and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine hydrochloride, a protein kinase inhibitor. DNA fragmentation was also inhibited by zinc ions, suggesting the involvement of a specific endonuclease in DNA cleavage. These phenomena are similar to those detected in thymocytes undergoing apoptosis following exposure to glucocorticoids (Cohen, J.J. and Duke, R.C. (1984) J. Immunol. 132, 38-42). Considering that topoisomerases function in cellular proliferation and differentiation by altering DNA topology, the results suggest that topoisomerases have important roles in T-lymphocyte ontogeny in the thymus and are in part involved in the elimination of autoreactive or harmful cells by an apoptotic process.