Several laboratories have earlier identified and characterized a specific prolactin binding site (receptor) on Nb2 cells which has a Kd of about 2 x 10(-10) M and a binding capacity of about 12,000 receptor sites per cell. Using [I125]-labeled human growth hormone (hGH), we have confirmed the results of these earlier reports. In addition, a second, "high-affinity" receptor site for prolactin has been identified. This site has a Kd of about 2 x 10(-12) M and there are an average of 67 sites per cell. Binding of [125]-hGH to the high-affinity receptor site is saturated by 1 min after incubation at 37 degrees C, whereas saturation of the lower affinity site requires a 1-3 hr incubation at 37 degrees C. Specificity of prolactin binding to both the high- and low-affinity "receptors" was established by the fact that several nonlactogenic hormones, including bovine growth hormone and insulin, do not displace [I125]-hGH from the receptor sites. When Nb2 cells were incubated for 1 hr at 0 degrees C with 100 pg/ml [I125]-hGH, no binding to the high-affinity receptor was detected, thus demonstrating the temperature dependence of binding to this receptor. The kinetics of binding of hGH to the high-affinity binding site correlates well with the dose-response effects of lactogenic hormones on mitogenesis in the Nb2 cells. In addition, the rapid binding of hGH to the high-affinity receptor may be related to specific effects of lactogenic-hormones which occur within seconds or minutes after adding these hormones to Nb2 cells.