Suspensions of rat hepatocytes isolated enzymatically by the method of Berry and Friend were used to study the binding of 125I-labeled human (hGH) and bovine (bGH) growth hormones and ovine prolactin (oPRL). Displacement of these labeled hormones by their unlabeled analogues was analyzed by means of Scatchard plots and affinity constants (K) and the number of binding sites per cell (q) were calculated. Specificity of binding was studied using hGH, bGH oPRL and rat growth hormone (rGH) and rat prolactin (rPRL). Rat hepatocytes contained two types of binding sites which bound hGH. The first, somatogenic, was specific for the growth-promoting hormones bGH and rGH. The second, lactogenic, was specific for lactogenic hormones, oPRL and rPRL. Human GH, which has both lactogenic and growth-promoting properties in rodents, bound to both sites. The somatogenic binding sites were present in both males and females, and the number of sites was similar in females and in males and was not affected by hypophysectomy. The lactogenic binding sites were present only in females, and the number of lactogenic and somatogenic sites was similar (40,000/cell). The affinity of hGH for the lactogenic binding sites was less than for the somatogenic (0.37 X 10(9) vs. 1 X 10(9)M-1). The lactogenic binding sites were lost when female rats were hypophysectomized and could not be restored by estrogen treatment.