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 mu-agonists. Herkinorin (HERK), unlike DAMGO, does not recruit beta-arrestin and promote mu-receptor internalization, even in cells that over express beta-arrestin. We hypothesized that chronic HERK and DAMGO treatment will differentially affect cellular markers of tolerance and dependence. CHO cells expressing the cloned human mu-receptor were treated for 20 h with 10 microM DAMGO, HERK, morphine, or medium. Both DAMGO and HERK acted as full agonists in the [(35)S]GTP-gamma-S binding assay with E(MAX) values of 230% and EC(50) values of 12.8 and 92.5 nM, respectively. In the cAMP assay, DAMGO and HERK had similar E(MAX) values of approximately 80% and EC(50) values of 3.23 and 48.7 nM, respectively. Chronic exposure to both drugs produced moderate tolerance to both drugs ( approximately 2 to 5 fold) in the [(35)S]GTP-gamma-S binding assay. In the cAMP assay, chronic DAMGO produced tolerance to both drugs ( approximately 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 mu-agonists produce cellular signs of tolerance and dependence.