The regulation of FSHbeta messenger RNA (mRNA) expression is complex and involves signals from the hypothalamus and gonads. Additionally, the local (pituitary) production of activin and follistatin appears to serve as an important modulator of endocrine signals for FSHbeta regulation. The purpose of these studies was to identify factors controlling pituitary activin/inhibin subunit and follistatin mRNA production in male and female rats. Both males and females expressed the follistatin, inhibin alpha, and betaB mRNAs, whereas the betaA mRNA was not detected. In males, levels of FSHbeta and follistatin were higher than those in females. After gonadectomy, levels of FSHbeta and follistatin increased in both sexes, whereas betaB rose only in females. In males, blockade of GnRH action from the time of castration prevented the increase in FSHbeta and follistatin, suggesting that GnRH is the primary stimulus for these gene products. In females, treatment with a GnRH antagonist only partially prevented the rise in FSHbeta, follistatin, and betaB expression, suggesting that other factors were also important. Passive immunoneutralization of circulating inhibin increased FSHbeta and follistatin (but not betaB), providing evidence that inhibin is a physiological regulator of follistatin. Replacement of estradiol at the time of ovariectomy prevented the increase in betaB mRNA, suggesting that gonadal steroids may also act via local factors to regulate FSHbeta. In summary, these studies provide evidence that GnRH, gonadal steroids, and gonadal peptides probably regulate FSHbeta expression at least in part via the intrapituitary activin/follistatin system.