We tested the hypotheses that the carboxylate side chain of Asp147 of the mu opioid receptor interacts with the protonated nitrogen of naltrexone and morphine and that this interaction is important for pharmacological properties of the two compounds. Mutation of Asp147 to Ala or Asn substantially reduced the affinity of naltrexone and the affinity, potency and efficacy of morphine, while the Glu mutant had similar properties as the wildtype, indicating the significant role of the carboxylate group of Asp147 in receptor binding and activation. This role could be due to its direct interaction with ligands or involvement in interhelical interactions. The unprotonated analogs of naltrexone and morphine, cyclopropylcarbonyl noroxymorphone (CPCNOM) and N-formylnormorphine (NFNM), respectively, were used to discriminate between these mechanisms. CPCNOM was much less potent as an antagonist and had substantially lower affinity for the mu receptor than naltrexone. Similarly, NFNM was unable to activate the mu receptor and had much lower affinity than morphine. These results indicate the importance of the protonated nitrogen. Notably, the D147A and D147N mutations did not appreciably affect the binding affinities of CPCNOM and NFNM. In addition, the D147E mutant had similar affinities for CPCNOM and NFNM as the D147A and D147N mu receptors. Thus, the carboxylate group of Asp147 is not important for binding of the two unprotonated compounds. These results indicate that the carboxylate group of Asp147 of the mu receptor interacts directly with the protonated nitrogen of naltrexone and morphine and this interaction is important for binding and receptor activation.