The nature of purported receptors for oxytocin (OT) in the rat adenohypophysis was investigated by means of radioligand binding. Tritium-labeled oxytocin (3H-OT) was used as the radioligand. Particulate fractions prepared from the adenohypophysis of estrogen-treated female rats bound 3H-OT reversibly and with high affinity (Kd = 1.5 nM). Scatchard plots indicated the presence of a single, low-capacity binding-site (Bmax = 33 fmol/mg protein). However, ligand displacement experiments suggested that 3H-OT labels OT- as well as vasopressin-preferring sites in adenohypophysial membrane-suspensions. When the number of pituitary vasopressin receptors was reduced by adrenalectomy, it was revealed that the ligand-specificity of the high-affinity OT binding site is similar to that of classical OT receptors. The results show the presence of separate receptors for OT and vasopressin in the rat adenohypophysis, and hence reinforce the notion that OT may function as a hypophysiotrophic hormone.