Growth of HeLa S3 cells in monolayer cultures of Joklik's minimum essential medium, 5% fetal calf serum, and 2 mM glutamine in the presence of 10(-6) M dexamethasone results in an approximately 70% inhibition of exogenously added [3H]thymidine incorporation into DNA. In marked contrast, dexamethasone does not alter HeLa S3 cell growth rate under these conditions. The half-maximal inhibitory concentration of dexamethasone is approximately 10(-9) M which correlates well with the dissociation constant of the nuclear glucocorticoid receptor at 37 degrees. Only active glucocorticoids, e.g., dexamethasone and cortisol, inhibit [3H]thymidine incorporation into DNA. 17 beta-Estradiol, 5 alpha-dihydrotestosterone, progesterone, and cortisone are ineffective. A measurable effect of dexamethasone at 10(-8) M occurs within 3 to 4 hr after hormone administration. The presence of transcriptional and translational inhibitors during exposure of the HeLa S3 cells to glucocorticoids blocks the accumulation of the hormone effect. Dexamethasone has little or no effect on uridine and leucine incorporation into RNA and protein, respectively, under these conditions. These results demonstrate that the incorporation of a DNA precursor is regulated by glucocorticoid hormones in HeLa S3 cells. This effect is most likely mediated via an alteration in the thymidine precursor pool specific activity.