Prolactin binding in normal mammary glands and pregnancy-dependent mammary tumors and plasma prolactin levels were assayed near parturition in GR/A female mice. Mammary glands and tumors cut into 0.5-mm thick slices were incubated in Medium 199 containing 5 ng of 125I-labeled ovine prolactin at 37 degrees C for 2 hr, and specific prolactin binding was expressed in terms of the difference between total and nonspecific bindings per mg tissue. On day 19 of pregnancy, prolactin binding was low in both normal mammary glands and tumors. One day after parturition, the binding of nonlactating mammary gland was as low as that on day 19 of pregnancy. On the other hand, the binding of lactating glands was raised significantly. There was no difference in the binding of tumors between lactating and nonlactating animals and both were significantly higher on day 1 after parturition than on day 19 of pregnancy. The plasma prolactin level assayed by radioimmunoassay was significantly higher on day 19 of pregnancy than on day 1 after parturition regardless of lactation. No relations were observed between the plasma prolactin level and the prolactin binding of either normal or neoplastic mammary glands.