Phytoestrogens, plant derived estrogen like-compounds exert numerous effects on the reproductive functions of animals. The present study was designed to demonstrate if exogenous genistein infused during the breeding season into the third ventricle of the brain of ovariectomized ewes could affect the secretory activity of the GnRH/LH axis. Two-year-old ovariectomized ewes (n=8) were infused with vehicle (control, n=3) or genistein (10 microg/100 microl/h, n=5) into the third ventricle. The infusions were done from 10.00 to 14.00 h and blood samples collection was performed this day up to 20.00 h and next day from 8.00 to 10.00 h. The animals were slaughtered, thereafter. Immunoreactive (IR) GnRH neurons in the hypothalamus and LH cells in the adenohypophysis were localized by immunohistochemistry. Messenger RNA analyses were performed by nonisotope in situ hybridization using sense and anti-sense riboprobes produced from beta subunits of LH cDNA clones. Plasma LH concentrations were measured by radioimmunoassay. Immunohistochemical analysis revealed that genistein infusion affected the morphology of GnRH neurons evoking a visualization of long axons in the GnRH perikarya and visibly diminished IR GnRH stores in the median eminence. The number of IR LH cells and IR material stored in the adenohypophyses increased in genistein-infused animals, which was confirmed by statistical analysis (P<0.001). The in situ hybridization analyses showed in these ewes the increase of mRNA LHbeta hybridization signal. The changes in LH release in response to genistein infusion had a biphasic character: it decreased within 6 h after infusion and increased 24 h later. Mean concentration of LH and amplitude of pulses measured from the beginning of infusion up to end of the experiment were significantly higher (P<0.05) in genistein-infused ewes compared to vehicle-treatment. In conclusion, our data show that genistein, a phytoestrogen, may effectively modulate GnRH and LH secretion in OVX ewes by acting directly on the CNS. The biphasic character of the LH response is similar to that of estradiol during the breeding season in the ewes.