A system has been developed for long-term incubation of rat thymus cells in sus pension in a chemically defined medium consisting of medium 199 and methylcellulsoe. With this system it is possible to study quantitatively the full time-course of glucocorticoid actions, from the early inhibitory effects on glucose uptake to the slow cytolytic effects. Cells incubated under these conditions preserve their sensitivity to the inhibitory effects of cortisol on glucose uptake for al least 12 h. Glucocorticoids at physiological concentrations lead to cytolysis (measured by reduction in viable cell counts determined by a modification of the pronase-cetrimide method) by 12 h incubation time. The cytolytic effect exhibits well-defined dose-response relationships and specificity for glucocorticoids. By 24 h,cortisol at 10(-6)M consistently reduces the total cell count by about 15%, and the viable cell count by about 40%. Cortisol is active down to 10(-7) M. Dexamethasone is roughly 10 times as active as cortisol, and cortisone completely inactive. Cortexolone is slightly active, but also shows antiglucocorticoid activity by its ability to block the action of cortisol. Under anerobic conditions in Medium 199, no inhibitory effect of cortisol is observed on glucose uptake.