Thyrostimulin is a heterodimeric hormone comprised of two glycoprotein hormone subunits, namely glycoprotein hormone subunit alpha2 and glycoprotein hormone subunit beta5 (GPB5). Immunological studies have revealed that both subunits colocalize in human pituitary corticotroph cells. Although recombinant thyrostimulin protein selectively activates the TSH receptor and has thyrotropic activity in rats, its biological functions have not been clarified. To explore the physiological regulators for the GPB5, the 5'-flanking region of the GPB5 coding sequence up to 3-kb upstream was analyzed by luciferase reporter assays. We found that nuclear factor-kappaB (NF-kappaB) markedly activated GPB5 transcription. Disruption of the putative NF-kappaB-binding motifs in the GPB5 5'-flanking region silenced the GPB5 activation by p65. Chromatin immunoprecipitation assays revealed that recombinant p65 bound to the predicted NF-kappaB-binding sites. Because NF-kappaB is known to associate with acute phase inflammatory cytokines, we examined whether TNFalpha or IL-1beta could regulate GPB5. Both these cytokines activated GPB5 transcription by 2- to 3-fold, and their effects were abolished by the addition of MG132, a NF-kappaB inhibitor. Our results suggest that inflammatory cytokines positively regulate thyrostimulin through NF-kappaB activation.