In addition to its role in causing Müllerian duct regression, Müllerian inhibiting substance (MIS) is implicated in the regulation of steroidogenesis, breast and prostate growth, and ovarian follicle recruitment, all of which are processes controlled or influenced by the hypothalamic-pituitary-gonadal axis. Whereas the direct effect of MIS on gonadal, prostate, and breast cells is under investigation, the ability of MIS to modulate pituitary function, thereby affecting those tissues indirectly, has not yet been studied. Using LbetaT2 cells, a murine gonadotrope-derived cell line, we have evaluated the effects of MIS on the expression of the gonadotropin genes. We show that both LbetaT2 cells and adult rat pituitaries express MIS type II receptor (MISRII) mRNA. Within 2 h, follicle-stimulating hormone beta subunit (FSHbeta) mRNA levels are significantly induced by addition of MIS to LbetaT2 cells and remain elevated through 8 h of treatment. Transcriptional activation of both the FSHbeta and luteinizing hormone beta subunit (LHbeta) gene promoters was observed by MIS, which enhances the effect of gonadotropin-releasing hormone (GnRH) agonist on the FSHbeta gene promoter and synergizes with the GnRH agonist to stimulate LHbeta gene promoter activity. Addition of MIS to LbetaT2 cells stimulates the activity of the rat LHbeta gene promoter with as little as 1 microg/ml and in a dose-dependent manner. These studies report both MISRII expression in rat pituitary cells and a gonadotrope-derived cell line and MIS-mediated activation of LHbeta and FSHbeta gene expression, and suggest that MIS may modulate the hypothalamic-pituitary-gonadal axis at more than one level.