Mammary epithelial cells isolated from midpregnant mice and cultured on collagen gels contain soluble glucocorticoid receptors. The kinetics of binding of dexamethasone reveal a saturable binding site [dissociation constant (Kd), approximately 1 nM], and the binding site obeys a steroid specificity characteristic of a glucocorticoid receptor. As with the receptor isolated from intact glands, the receptor from the cultured cells also requires the addition of dithiothreitol for maximal binding of dexamethasone. The receptors are maintained at in vivo levels (approximately 1.3 pmol/mg DNA) for at least a period of 10 days in culture. However, the presence of both cortisol and PRL is required for the maintenance of the receptors, and the effect of both these hormones is dose dependent.