Tumors formed from wild type P1798 mouse lymphoma cells undergo regression when treated with pharmacological doses of natural and synthetic glucocorticoids in vivo. Variants have been selected that are insensitive to the cytolytic effects of glucocorticoids in vivo. Although the response of wild type and insensitive tumors is markedly different in vivo, the manner in which cells from such tumors respond to glucocorticoids is indistinguishable in culture under routine conditions. Glucocorticoids inhibit proliferation of wild type cells as well as those that are insensitive to glucocorticoids in vivo. Although neither cell line dies when exposed to dexamethasone in culture in the presence of fetal bovine serum, both sensitive and insensitive cell lines undergo cytolysis when exposed to dexamethasone in serum-free medium. Sensitive cells die more quickly, with 50% cell death observed within 6 h. Insensitive cells exhibit <10% cell death within 6 h. Sensitive cells continue to die after transitory exposure to dexamethasone, whereas insensitive cells do not. Thus, growth in serum-free medium mimics the response that prevails in vivo. Cell death is associated with rapid, internucleosomal chromatin degradation. The rate of DNA fragmentation is comparable to that of cell death. About 30% of the DNA in sensitive cells is degraded to fragments of <10 kilobases within 2 h after addition of dexamethasone, and 70–80% of the DNA is degraded within 6 h. There is no significant degradation observed when insensitive cells are treated for 6 h. P1798 cell lines express an endonuclease that is capable of degrading chromatin in vitro. Basal expression of this activity does not correlate with glucocorticoid sensitivity, and insensitivity does not appear to be attributable for glucocorticoid-mediated chromatin degradation. The data suggest that glucocorticoid insensitivity is associated with delayed activation and/or induction of some lytic principle. Alternatively, resistance may be due to enhanced ability to repair the damage induced by transitory exposure to glucocorticoids in vivo.