The ability of a series of specific Galpha carboxyl-terminal antisera, (i.e. anti-Gsalpha, anti-Gi1/2alpha, anti-Gi3alpha/Goalpha, anti-Goalpha/Gi3alpha, and anti-Gq/11alpha) to disrupt (+/-)-baclofen-stimulated high-affinity guanosine triphosphatase (GTPase) activity was explored in rat cerebral cortical membranes to identify the Galpha subunit(s) involved in gamma-aminobutyric acid(B) (GABA(B)) receptor-mediated signal transduction. Pretreatment of the membranes with the AS/7 (anti-Gi1/2alpha) antiserum inhibited GABA(B) receptor-mediated response without affecting the basal activity. The RM/1 (anti-Gsalpha) and QL (anti-Gq/11alpha) antisera failed to inhibit GABA(B) receptor-coupled responses. The results of the EC/2 (anti-Gi3alpha/Goalpha) and GO/1 (anti-Goalpha/Gi3alpha) antisera were difficult to interpret since the basal activities were influenced by these antisera. These results, in conjunction with the data in our previous reconstitution study, indicate that Gi2alpha is a main transducer of GABA(B) receptor-mediated signaling in rat cerebral cortex.