Regulation of mammary prolactin receptors by steroid hormones was investigated in ovariectomized mid-pregnant mice. Ovariectomy increased the number of mammary prolactin receptors per cell with no effect or a slight decrease in dissociation constant (Kd). The simultaneous removal of adrenals prevented this increase in numbers. A single injection of glucocorticoid (corticosterone or cortisol) in ovariectomized–adrenalectomized mice restored the number of prolactin receptors in mammary glands to the same level as that in ovariectomized controls without changing the Kd. Aldosterone, deoxycorticosterone and oestradiol did not affect the number of mammary prolactin receptors after ovariectomy–adrenalectomy. Serum concentration of prolactin was not influenced by the hormone manipulation except with injections of oestradiol or cortisol and apparently did not correlate with the number of prolactin receptors. These results indicated that glucocorticoids are required for the increase in the number of mammary prolactin receptors induced by ovariectomy in mid-pregnant mice.