The G-protein activation induced by mu-opioid receptor agonists in the pons/medulla membrane obtained from mice lacking exons 2 and 3 of mu-opioid receptor gene (MOR (Exons 2 and 3)-knockout (KO) mice) was investigated by monitoring guanosine-5'-o-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. The MOR agonists D-Ala2,MePhe4,Gly(ol)5)enkephalin, endomorphin-1 and endomorphin-2 each produced concentration-dependent increases in [(35)S]GTPgammaS binding to pons/medulla membrane in wild-type mice, but not in MOR (Exons 2 and 3)-KO mice. beta-Endorphin also produced a concentration-dependent increase of [(35)S]GTPgammaS binding to pons/medulla membrane in wild-type mice, however the increase of [(35)S]GTPgammaS binding induced by beta-endorphin was partially attenuated in MOR (Exons 2 and 3)-KO mice. The present results suggest that MOR that is created from the sequences encoded with exons 2 and 3 of the MOR gene, as has been previously observed in studies of mice lacking exon 1 of this gene, may be another critical target for the activation of G-protein by MOR agonists in the mouse pons/medulla.