In the pituitary, GABA regulates the release of several hormones via different receptors. GABA(C) receptors are heterooligomers that differ from GABA(A) receptors in that they contain p-subunits and are insensitive to bicuculline. However, molecular and functional evidence for the presence of GABA(C) receptors outside the retina has yet to be established. The present work was performed on guinea pig and rat pituitaries. Both Northern blot and RT-PCR analysis showed that, although rho1- and rho2-subunits were expressed at similar levels in the rat retina, rho1 messenger RNA (mRNA) was enriched, relative to rho2 mRNA in the rat pituitary. Northern blot experiments also showed that, in the pituitary, rho1 and rho2 mRNAs are shorter in size than those expressed in the retina. The use of a subunit-specific antibody revealed colocalization of rho1-subunit and anti-TSH labeling on rat pituitary sections. TSH guinea pig pituitary cells were also labeled with a rho-subunit antiserum. Moreover, whole-cell patch clamp on single guinea pig TSH cells showed that GABA induced a bicuculline-insensitive Cl- current. In contrast to the Cl- current generated by GABA(C) receptors in the retina, the bicuculline-insensitive Cl- currents in TSH cells quickly desensitized. These results suggest that a novel GABA(C) receptor may regulate TSH secretion and that the structure and/or biochemical regulation of this pituitary receptor is different from that found in the retina.