Background and Purpose: Adverse side effects of conventional opioids can be avoided if ligands selectively activate peripheral opioid receptors in injured tissue. Injury and inflammation are typically accompanied by acidification. In this study, we examined influences of low pH and mutation of the ionizable amino acid residue H2976.52 on μ-opioid receptor binding and signalling induced by the μ-opioid receptor ligands fentanyl, DAMGO, and naloxone. Experimental Approach: HEK 293 cells stably transfected with μ-opioid receptors were used to study opioid ligand binding, [35S]-GTPγS binding, and cAMP reduction at physiological and acidic pH. We used μ-opioid receptors mutated at H2976.52 to A (MOR-H2976.52A) to delineate ligand-specific interactions with H2976.52. Key Results: Low pH and the mutant receptor MOR-H2976.52A impaired naloxone binding and antagonism of cAMP reduction. In addition, DAMGO binding and G-protein activation were decreased under these conditions. Fentanyl-induced signalling was not influenced by pH and largely independent of H2976.52. Conclusions and Implications: Our investigations indicate that low pH selectively impairs μ-opioid receptor signalling modulated by ligands capable of forming hydrogen bonds with H2976.52. We propose that protonation of H2976.52 at acidic pH reduces binding and subsequent signalling of such ligands. Novel agonists targeting opioid receptors in injured tissue might benefit from lack of hydrogen bond formation with H2976.52.