Participation of central 5HT receptors in the inhibition of LH pulsatility during refractoriness to short days (SD) in ewes has been suggested by previous in vivo studies using various 5HT-antagonist such as ketanserin. In the present study, binding of [<sup>3</sup>H]ketanserin in ewe brain sections was similar to that described in the brain of other species and could correspond with an interaction at 5HT2 receptors sites. Rosenthal analysis from the caudate nucleus was linear (Kd = 3 n M). The displacement studies from the cortex slices showed that the 5HT antagonists such as methysergide, ketanserin, cyproheptadine and spiperone competed with the labelled ligand at nanomolar concentrations whereas serotonin was less active. However, the first 3 drugs recognized different populations of binding sites. Prazosin, an α<sub>1</sub>-adrenergic antagonist was inactive, but a slight inhibition of [<sup>3</sup>H]ketanserin binding was induced by pyrilamine, an H<sub>1</sub> histaminic antagonist, within a nanomolar range. Methysergide (10<sup>–6</sup> M), which does not bind to H<sub>1</sub> receptors, was therefore used to determine the nonspecific binding. Quantitative analysis of the binding of 3 n M [<sup>3</sup>H]ketanserin on sections of the ewe brain at the preopticohypothalamic level was then carried out by autoradiography. The highest binding densities were observed in the caudate nuclei (64.0 fmol/mg tissue Eq) and the mammillary bodies (52.7 fmol/mg tissue Eq) whereas intermediate or low densities were found in the other structures. The anatomical distribution of the labelling was similar to that described in other species for 5HT<sub>2</sub> receptors. Ketanserin binding in these areas was compared between two groups of ovariectomized estradiol-treated Ile-de-France ewes, submitted to artificial short days (SD: 8L: 16D), one group with a high LH pulsatility (responsive to SD) and the other one with a low LH pulsatility (photorefractory to SD). Binding densities were similar for each one of the studied regions between the two groups, except in the ventrolateral part of the mediobasal hypothalamus, where ewes exhibiting high LH pulsatility had a more than 2-fold higher binding density than those with a low LH pulsatility (mean ± SEM, 14.6 ± 1.4 vs. 5.7 ± 1.0 fmol/mg tissue Eq, respectively; p < 0.0016). These results suggest that [<sup>3</sup>H]ketanserin binding sites in the ventromedial part of the mediobasal hypothalamus could be associated to the regulation of the photoperiodic inhibition of LH at the time of establishment of refractoriness to short days in the Ile-de-France ewe.