Bovine mammary cells cultured in vitro were incubated with varying levels of 3H-cortisol and various unlabeled steroids. These mammary cells possessed 2 components which had a high affinity (Kd≍5×lO8 to 2×109M) for the uptake of cortisol. There were approximately 7500 specific binding sites per mammary cell. The incorporation of 3H-cortisol into the mammary cell is inhibited by many (but not all) corticoids, by progesterone, by 17α-OH-progesterone and by preheating to 100 C for 10 min. It is not inhibited by 17β-estradiol, testosterone or 20α-OH-progesterone. There was also a major nonspecific component which bound 3H-cortisol that was practically unsaturable and could not be destroyed by preheating to 100 C for 10 min. About 77% of the 3H-cortisol is found in the 105,000×g supernatant fluid with about 21% localized in the 700×g precipitate fraction of the cultured mammary cell. The majority of the cortisol incorporated into the cell remained as cortisol although metabolites were found in both the 700×g supernatant and precipitate fractions of the cell. The amount of 3H-cortisol which could be specifically inhibited varied according to the individual strain of mammary cells. During the first 30 days hi culture there was a loss in the ability of mammary cells to specifically incorporate 3H-cortisol, but after 30 days of culture there was no association between time in culture and cortisol binding ability. We conclude that bovine mammary cells cultured in vitro possess a specific mechanism for the binding of cortisol. (Endocrinology89: 152, 1971)