7,12-Dimethylbenz[alpha]anthracene-induced rat mammary tumors were dissociated with collagenase and hyaluronidase and placed into primary culture. In most cultures, specific binding of 125I-labeled ovine prolactin was (i) lower than that for the original tumors unless bovine prolactin (1 microgram/ml) had been added to the dissociation medium, and (ii) varied with the type of growth medium used. The level of prolactin binding in cultured cells was relatively constant for the first 7-10 days. Prolactin binding in cultured cell homogenates was maximal at pH 7.0, proportional to cell protein, specific for prolactin, and reached a steady state by 12 h at 22 degrees C. The half-maximum inhibition of 125I-labeled prolactin binding by unlabeled prolactin was 100 ng/ml for cells grown in 5-1000 ng of prolactin/ml. After prolactin was removed from the growth medium, the level of available binding sites progressively increased, reached a maximum at 48 h and then declined. At 48 h, the dissociation constant for prolactin binding (Kd approximately 1 x 10(-10) M) was comparable to that in tumors. In some cultured tumors, a 48-h treatment with 0.5 or 1.0 ng of prolactin/ml caused an apparent increase in the level of prolactin binding. Prolactin increased DNA synthesis and its removal caused a reduction in [3H]estradiol and [3H]-R5020 binding to cultured cell cytosols.