Glucocorticoid-induced lymphocytolysis has been studied for many years; however, the mechanism of lymphoid cell death has not been elucidated. In this study we have investigated the effects of glucocorticoids on the lymphocyte genome using the rat thymocyte model. Adrenalectomized rats were injected ip with dexamethasone (DEX) and killed thereafter. The thymus gland was removed, and DNA was extracted from isolated thymocytes and then separated electrophoretically on 1.8% agarose gels. Administration of glucocorticoids in vivo resulted in the cleavage of lymphocyte DNA at internucleosomal intervals. Genomic DNA separated on agarose gels from DEX-treated rats appeared as a ladder of DNA fragments which were multiples of about 180 base pairs, while DNA from control rats appeared as a single high mol wt band. This cleavage of thymocyte DNA was a rapid effect of adrenal steroid treatment and occurred before cell death. Thymocyte DNA fragmentation increased with time after DEX treatment and the dose of half-maximal response in vivo was estimated to be about 1.8 X 10(-8) M. Internucleosomal cleavage of DNA was only observed in lymphoid tissues (thymus and spleen), but not in other glucocorticoid-sensitive tissues (kidney, liver, heart, brain, or testis). Treatment of rats with estrogen, androgen, or progestin failed to elicit thymocyte DNA degradation. Glucocorticoid-induced DNA cleavage was partly inhibited by the glucocorticoid antagonist RU 486 (17 beta-hydroxy-11 beta,4-dimethylaminophenyl-17 alpha-propynl-estra-4,9-diene-3-one). These findings suggest that glucocorticoids activate, via a receptor-mediated process, an endonuclease-like activity in lymphoid tissues which cleaves the lymphocyte genome at internucleosomal sites. Activation of this nuclease by glucocorticoids precedes lymphocytolysis and may represent the hormonal regulation of programmed cell death.