This study was designed to test the hypothesis that genistein, a phytoestrogen, could immediately affect prolactin (PRL) and luteinizing hormone (LH) secretion in ovariectomized (OVX) ewes, when infused directly into the central nervous system (CNS) during short days. Starting 6 weeks after OVX, ewes were infused intracerebroventricularly (ICV) with a vehicle (control, n=5) or genistein (10 μg/100 μl/h, n=5). All infusions were performed from 12.00 to 16.00 h and blood samples were collected from 8.00 to 20.00 h at 10-min intervals. The plasma PRL and LH concentrations were assayed by the radioimmunoassay methods. In vehicle-infused ewes, the mean plasma PRL concentration decreased significantly (P<0.05) during the infusion, compared with the pre-infusion PRL value. In genistein-infused ewes, a significant (P<0.001) increase in the PRL concentration was observed during the infusion, compared with the values noted before and after the treatment. The PRL concentration recorded during genistein infusion was also significantly (P<0.001) higher than the concomitant concentration in vehicle-infused animals. The mean plasma LH concentration in vehicle-infused ewes was significantly (P<0.01) higher after than both before and during the infusion. In genistein-infused ewes, the LH concentration measured after was significantly lower than both before (P<0.001) and during the infusion (P<0.01). Significant (P<0.01) differences also occurred between the concomitant LH concentrations recorded in vehicle- and genistein-infused groups. These results demonstrate that genistein may affect the hypothalamo-pituitary neuroendocrine axes activity in long-term OVX ewes, under the conditions of short days.