Previous 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 efficacious μ-agonists. Herkinorin (HERK), unlike DAMGO, does not recruit β-arrestin and promote μ-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 human μ-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 with EMAX values of 230% and EC50 values of 12.8 and 92.5 nM, respectively. In the cAMP assay, DAMGO and HERK had similar EMAX values of ∼80% and EC50 values 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 noninternalizing μ-agonists produce cellular signs of tolerance and dependence. Synapse 61:166–175, 2007. Published 2006 Wiley-Liss, Inc.