Chimeric receptor studies have suggested that the binding and selectivity of the endogenous opioid peptide dynorphin A arise from the interaction of its "address" with the second extracellular loop (EL-2) of the κ-opioid receptor, and it is generally accepted that key basic residues in the dynorphin A "address" recognize EL-2 acidic residues through ionic interactions. However, modeling studies have suggested hydrophobic contacts may be involved. To experimentally evaluate the role of EL-2 anionic residues in dynorphin A binding and selectivity, we created a series of isosteric mutants where clustered negative charges in EL-2 were neutralized. Point mutations were introduced into the κ-opioid receptor, and mutants were transiently expressed in HEK cells for competitive binding experiments with [³H]diprenorphine and functional assays using the [³⁵S]GTPγS assay. Results showed that the remaining three mutants (E203QD204ND206N, E203QD204ND206NE209Q, D216ND217NE218Q) had Ki values for dynorphin A(1-13) comparable to the wild-type receptor, and their EC50 values in functional assays were also similar. Taken together, these data suggest that charge-charge interactions between EL-2 and the dynorphin "address" may not be as important as generally believed in determining dynorphin A's selectivity and binding affinity to the κ-opioid receptor. Hydrophobic interactions or other structural domains (e.g., the non-conserved Glu297 residue) may be involved in selective binding and activation.