The present study was designed to demonstrate whether genistein, a synthetic phytoestrogen, infused into the third ventricle of the brain could affect the gonadotrophic cells regarding the presence of oestrogen receptor-alpha immunoreactivity and gonadotrophin subunit mRNA hybridising reaction in the ewe. Ewes (n=7), aged 2 years, in early anoestrous season were infused with Ringer-Locke solution (control, n=3) or 10 microg/100 microl/h of genistein (n=4) into the third ventricle over a 5 h period and slaughtered the following morning. Immunoreactivity of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestrogen receptor-alpha (ERalpha) was determined in the adenohypophysis by immunohistochemistry using antibodies raised against LHbeta, FSHbeta, and ERalpha. Messenger RNA analyses were performed by non-isotope in situ hybridisation using sense and antisense riboprobes produced from beta subunits of LH and FSH cDNA clones. Computer image analysis was used to determine the percent of cells exhibiting immunohistochemical and/or hybridising reaction. It was found that in ewes infused with genistein, the percentage of LH-positive cells and the density of immunoreactive-LHbeta material decreased significantly (P<or=0.001), but the percentage of mRNA LHbeta-expressing cells and the intensity of the hybridisation signal increased significantly (P<or=0.001), compared to the vehicle-infused animals. The genistein infusions had no effect on the immunoreactivity of FSH cells or on the expression of mRNA for FSHbeta. The percentage of ERalpha-positive cells increased significantly after genistein infusions (P<or=0.001) and this increase was significant in the LH but not in FSH cells (P<or=0.001). In conclusion, we suggest that genistein can stimulate the expression of immunoreactive ERalpha in the pituitary LH-cells but not in FSH-cells and change the endocrine activity of LH-producing cells of anoestral ewes.