Corticosteroid-binding globulin (CBG) activity of rat serum was determined by multiple equilibrium dialysis using corticosterone-4- 14C at 37 C. Hypophysectomy did not significantly alter the CBG activity of male rats, but decreased markedly that of female animals. The maximum effect of hypophysectomy in the female rat was observed after 9 days and continued through the 24th day after operation; it was followed by a gradual lessening of the response. The injection of a rat anterior pituitary suspension into hypophysectomized female rats increased the CBG activity toward the level of the sham controls. The administration of either growth hormone, prolactin, luteinizing hormone or a human pituitary gonadotropin preparation did not alter the CBG activity when compared with that of hypophysectomized controls. Thyroid-stimulating hormone, however, enhanced the binding activity compared to that of sham-hypophysectomized animals. It was concluded that the hypophysial factor controlling CBG is the thyroid-stimulating hormone acting through increased secretion of thyroid hormone.