HeLa S3 cells contain high affinity, saturable protein receptor molecules which steriospecifically bind active glucocorticoids. The number of glucocorticoid receptors per cell changes as cells in culture proceed through the cell cycle (1). HeLa S3 cells brought to the GI/S boundary by the double thymidine block procedure undergo a rapid synchronous round of DNA synthesis when released into thymidine-free medium. Analysis of glucocorticoid receptor binding indicates a rapid rise in cellular receptor number when measurements are made in whole cells at either 3 C (cytoplasmic) or 37 C (nuclear). Cytoplasmic receptors are maintained at levels about 150% above late GI values throughout S and GII phases until mitosis occurs, whereas the nuclear binding of hormone is dramatically reduced during GII and remains low during mitosis and early GI. Similar cell cycle-dependent alterations in receptor number occur in synchronized cell populations obtained by unit gravity sedimentation. Sucrose density gradient analysis and Sephacryl S-200 gel filtration of cytoplasmic receptors during the cell cycle indicate a rapid increase in approximately 7-8S dexamethasone-binding component during early S phase, when receptors accumulate in cells. The role of RNA, protein, and DNA syntheses in mediating this S phase increase in receptor number were next examined. Both cycloheximide and puromycin blocked the increased receptors accumulation that occurs during the S phase, whereas delayed addition of these inhibitors led to partial increases in receptor binding. Several inhibitors of RNA synthesis [actinomycin D, L-521,818-00E10 (Merck), and alpha-amanitin] all effectively inhibited the S phase accumulation of receptor. Increased receptor binding during the S phase was not dependent on DNA replication. These data suggest that the increase in glucocorticoid receptor number during the S phase results from alterations in RNA and protein synthetic processes.