The present study was undertaken to investigate the potential role of glucocorticoid-induced DNA damage in the lysis of human lymphoid leukemia cells by glucocorticoids. Lymphoblasts were isolated from patients with acute lymphoblastic leukemia (ALL) or chronic myelogenous leukemia (CML) in blast crisis and cultured in vitro with or without dexamethasone. DNA was then purified from the cells and analyzed by agarose gel electrophoresis. Only high molecular weight (mol wt) DNA was present in cells cultured without dexamethasone, but a ladder of DNA fragments ranging in size from 180 to 200 base pairs (bp) to greater than 1,500 bp was present in cells cultured with dexamethasone. The DNA fragments were multiples of 180 to 200 bp, suggesting an internucleosomal site of DNA cleavage. The same pattern of DNA fragmentation was detected in normal thymocytes isolated from adrenalectomized rats following in vivo treatment with dexamethasone and in S49 mouse thymoma cells after in vitro incubation with dexamethasone. Dexamethasone-induced DNA fragmentation preceded overt loss of viability in glucocorticoid-sensitive cells but was not detected in two variants of the S49 cell line that are glucocorticoid resistant owing to glucocorticoid receptor defects. The results suggest that glucocorticoids kill human lymphoblastic leukemia cells and both normal and malignant murine thymocytes by a common mechanism that involves glucocorticoid induction of an endonucleolytic activity with cleavage of genomic DNA.