Serum prolactin concentration was studied in hemiovariectomized rats, hemiovariectomized rats bearing chronic estradiol benzoate (E2) implant either in the anterior (A-AHA) or in the middle (M-AHA) portion of the anterior hypothalamic area (AHA), and was also studied in ovariectomized AHA lesioned rats. Hemiovariectomy caused rise of serum porlactin which peaked at day 10 and thereafter declined to the basal level. E2 implanted in the M-AHA either unilaterally or bilaterally temporarily suppressed the serum porlactin rise induced by hemiovariectomy. Unilateral implant of E2 in the A-AHA elicited also a temporary but longer suppressive effect. Bilateral E2 implant in the A-AHA consistently suppressed serum prolactin of hemiovariectomized animals. Ovariectomized AHA lesioned rats responded to systemic E2 treatment with a slight increase of serum prolactin while the non-lesioned ovariectomized rats responded with a significant increase. The additional E2 treatment effectively enhanced serum prolactin elevation in AHA ablated animals. The results substantiated the involvement of the AHA in prolactin secretion as well as they disclosed the A-AHA as a dominant area while the M-AHA appears to be supplementary area for prolactin inhibition, provided that the areas were in response to E2 implantation.