In order to better understand the cellular mechanisms underlying LH and FSH secretion, we have addressed the contribution of lipid rafts to the secretion of gonadotropins. We used methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering agent, on an LbetaT2 murine gonadotroph cell line and on primary cultures of ovine pituitary cells. We found that in both systems, cholesterol depletion by MbetaCD induced a fast and substantial release of LH in the absence of natural stimulation by GnRH. In ovine pituitary cells, MbetaCD-mediated LH release was shown to be independent of protein synthesis. Twenty-four hours after MbetaCD treatment, there was no loss of cell viability and full recovery of LH secretory capabilities, as determined by GnRH or MbetaCD treatment. In addition, our data suggest the existence of a pool of LH that is not released by GnRH treatment but that is released by MbetaCD treatment. Finally, in ovine pituitary cells, MbetaCD treatment induced FSH secretion. Importantly, these in vitro data are supported by in vivo studies, because MbetaCD injected into the pituitary glands of anaesthetized sheep reproducibly induced a peak of LH release.