AbstractPrevious studies established that Tyr‐D‐Ala‐Gly‐N‐Me‐Phe‐Gly‐ol (DAMGO) and (2S,4aR,6aR,7R,9S,10aS,10bR)‐9‐(Benzoyloxy)‐2‐(3‐furanyl)dodecahydro‐6a,10b‐dimethyl‐4,10‐dioxo‐2H‐naphtho‐[2,1‐c]pyran‐7‐carboxylic acid methyl ester (herkinorin) are fully efficaciousμ‐agonists. Herkinorin (HERK), unlike DAMGO, does not recruit β‐arrestin and promoteμ‐receptor internalization, even in cells that over express β‐arrestin. We hypothesized that chronic HERK and DAMGO treatment will differentially affect cellular markers of tolerance and dependence. CHO cells expressing the cloned humanμ‐receptor were treated for 20 h with 10 μM DAMGO, HERK, morphine, or medium. Both DAMGO and HERK acted as full agonists in the [35S]GTP‐γ‐S binding assay withEMAXvalues of 230% and EC50values of 12.8 and 92.5 nM, respectively. In the cAMP assay, DAMGO and HERK had similarEMAXvalues of ∼80% and EC50values of 3.23 and 48.7 nM, respectively. Chronic exposure to both drugs produced moderate tolerance to both drugs (∼2 to 5 fold) in the [35S]GTP‐γ‐S binding assay. In the cAMP assay, chronic DAMGO produced tolerance to both drugs (∼3 to 4 fold). Chronic HERK eliminated the ability of either drug to inhibit forskolin‐stimulated cAMP accumulation. Chronic DAMGO increased, and chronic HERK decreased, forskolin‐stimulated cAMP accumulation. Naloxone, after chronic HERK (but not DAMGO) induced a large increase in forskolin‐stimulated cAMP accumulation. Viewed collectively with published data, the current data indicate that both internalizing and noninternalizingμ‐agonists produce cellular signs of tolerance and dependence. Synapse 61:166–175, 2007. Published 2006 Wiley‐Liss, Inc.