The G-protein activation induced by μ-opioid receptor agonists was determined in spinal cord membranes from two types of μ-opioid receptor knockout mice: mice with a disruption of exon 1 (MOR (Exon 1)-KO) or exons 2 and 3 (MOR (Exons 2 and 3)-KO) of the μ-opioid receptor gene. The G-protein activation induced by the opioid agonists was measured by monitoring the increases of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding. The μ-opioid receptor agonists (D-Ala2,N-MePhe 4,Gly-ol5]enkephalin, endomorphin-1, endomorphin-2, morphine, morphine-6β-glucuronide, and fentanyl produced concentration-dependent increases of [35S]GTPγS binding to spinal cord membranes in wild-type mice, but not in MOR (Exon 1)-KO mice or MOR (Exons 2 and 3)-KO mice. On the other hand, the δ-opioid receptor agonist [D-Pen 2,5]enkephalin, the κ-opioid receptor agonist (−)U50,488H, or the ORL1-receptor agonist nociception increased [35S]GTPγS binding in the spinal cord membranes from both MOR (Exon 1)-KO mice and MOR (Exons 2 and 3)-KO mice to the same extent as in the corresponding wild-type mice. The results provide further information about the important roles of the sequences encoded within exon 1 and exons 2 and 3 of μ-opioid receptor gene for the activation of G-proteins by μ-opioid receptor agonists in the mouse spinal cord.