Opioid analgesics constitute the most effective treatment of severe pain, but they also cause adverse and often fatal side effects. Selective targeting of peripheral opioid receptors in injured tissue is a promising strategy to convey analgesia devoid of severe adverse effects. Drug targeting to injured tissue can be achieved by exploiting the acidic pH associated with inflammation. Yet, little is known on ligand recognition and signaling of opioid receptors at acidic pH. In the present study, I demonstrate that at acidic pH, μ-opioid receptor (MOR) responses are modulated depending on a specific structural property of bound ligands. In radioligand binding experiments, acidic pH reduced binding of naloxone (NLX) and [D-Ala2,N-Me- Phe4,Gly5-ol]-enkephalin (DAMGO), ligands that form hydrogen bond networks to histidine residue H297/6.52 within the receptor binding pocket. Furthermore, I observed impaired DAMGO-induced G-protein activation (as assessed by [35S]-GTPγS binding) and naloxone modulation of cAMP levels at acidic pH. In contrast, acidic pH did not alter G-protein activation and cAMP responses induced by fentanyl, a ligand that is unable to form hydrogen bonds to MOR residue H297/6.52. The exchange of residue H297/6.52 by alanine (A) abolished high-affinity binding of [3H]-NLX and [3H]-DAMGO, as well as DAMGO-induced G-protein activation. This mutation did not significantly alter fentanyl-induced Gprotein activation, but reduced cAMP responses to fentanyl. I conclude that H297/6.52A is crucial to effective binding of DAMGO and NLX to the MOR, while this residue contributes less to the binding of fentanyl. Overall, these findings indicate that acidic pH selectively impairs binding and consequent signaling of ligands that strongly depend on hydrogen bond formation to H297/6.52. To ensure maximum MOR binding and signaling in inflamed tissue, I suggest that opioid ligands should bind largely independent of H297/6.52.