Background and PurposeThis study examined the role of agents known to activatePKCon morphine‐induced desensitization of μ‐opioid receptors (MOPreceptors) in brain slices containing locus coeruleus neurons.Experimental ApproachIntracellular recordings were obtained from rat locus coeruleus neurons. Two measurements were used to characterize desensitization, the decline in hyperpolarization induced by application of a saturating concentration of agonist (acute desensitization) and the decrease in hyperpolarization induced by a subsaturating concentration of [Met]5enkephalin (ME) following washout of the saturating concentration (sustained desensitization). Internalization ofMOPreceptors was studied in brain slices prepared from transgenic mice expressingFlag‐MOPreceptors. The subcellular distribution of activatedPKCwas examined using a novel fluorescent sensor ofPKCinHEK293 cells.Key ResultsThe phorbol esters (PMA and PDBu) and muscarine increased acute desensitization induced by a saturating concentration of morphine andME. These effects were not sensitive to staurosporine. Staurosporine did not block the decline in hyperpolarization induced by muscarine. PDBu and muscarine did not affect sustained desensitization induced byMEnor did phorbol esters or muscarine change the trafficking ofMOPreceptors induced by morphine orME. The distribution of activatedPKCmeasured inHEK293 cells differed depending on which phorbol ester was applied.Conclusions and ImplicationsThis study demonstrates a distinct difference in two measurements that are often used to evaluate desensitization. The measure of decline correlated well with the reduction in peak amplitudes caused byPKCactivators implicating the modification of other factors rather thanMOPreceptors.Linked ArticlesThis article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visithttp://dx.doi.org/10.1111/bph.2015.172.issue-2