We examined whether a proposed spatial proximity between Asp114(2.50) and Asn332(7.49) affected the functional properties of the mu opioid receptor. The D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO and CTAP, but not for naloxone and [3H]diprenorphine; this mutation also abolished agonist-induced increase in [35S]GTPgammaS binding. The N332(7.49)D mutation eliminated detectable binding of either [3H]diprenorphine or [3H]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affinity binding for [3H]diprenorphine, CTAP and naloxone, and restored partially the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the residues are adjacent in space and that their chemical functionalities are important for ligand binding and receptor activation.