AbstractIn morphine tolerance a key question that remains to be answered is whether μ‐opioid receptor (MOPr) desensitization contributes to morphine tolerance, and if so by what cellular mechanisms. Here we demonstrate that MOPr desensitization can be observed in single rat brainstem locus coeruleus (LC) neurons following either prolonged (> 4 h) exposure to morphinein vitroor following treatment of animals with morphinein vivofor 3 days. Analysis of receptor function by an operational model indicated that with either treatment morphine could induce a profound degree (70–80%) of loss of receptor function. Ongoing PKC activity in the MOPr‐expressing neurons themselves, primarily by PKCα, was required to maintain morphine‐induced MOPr desensitization, because exposure to PKC inhibitors for only the last 30–50 min of exposure to morphine reduced the MOPr desensitization that was induced bothin vitroandin vivo. The presence of morphine was also required for maintenance of desensitization, as washout of morphine for > 2 h reversed MOPr desensitization. MOPr desensitization was homologous, as there was no change in α2‐adrenoceptor or ORL1 receptor function. These results demonstrate that prolonged morphine treatment induces extensive homologous desensitization of MOPrs in mature neurons, that this desensitization has a significant PKC‐dependent component and that this desensitization underlies the maintenance of morphine tolerance.