The identity of the G-protein coupling thyrotropin-releasing hormone (TRH) receptors to rat ether-a-go-go related gene (r-ERG) K⁺ channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH₃ cells and dominant-negative variants (G-QL/DN) of G-protein subunits. Expression of dominant-negative G_q/11 that minimizes the TRH-induced Ca²⁺ signal had no effect on r-ERG current inhibition elicited by the hormone. In contrast, the introduction of dominant-negative variants of G₁₃ and the small G-protein Rho caused a significant loss of the inhibitory effect of TRH on r-ERG. A strong reduction of this TRH effect was also obtained in cells expressing either dominant-negative G_s or transducin subunits, an agent known to sequester free G-protein dimers. As a further indication of specificity of the dominant-negative effects, only the dominant-negative variants of G₁₃ and Rho (but not G_s-QL/DN or G_t) were able to reduce the TRH-induced shifts of human ERG (HERG) activation voltage dependence in HEK293 cells permanently expressing HERG channels and TRH receptors. Our results demonstrate that whereas the TRH receptor uses a G_q/11 protein for transducing the Ca²⁺ signal during the initial response to TRH, this G-protein is not involved in the TRH-induced inhibition of endogenous r-ERG currents in pituitary cells. They also identify G_s (or a G_s-like protein) and G₁₃ as important contributors to the hormonal effect in these cells and suggest that dimers released from these proteins may participate in modulation of ERG currents triggered by TRH.