Several cellular systems display desensitization and downregulation of opioid receptors upon chronic treatment, suggesting that they could be used as a model system to understand opioid tolerance/dependence. However, a model system containing a homogeneous population of mu-opioid receptors, the receptors at which morphine and related opioids act, has been lacking. To approach this problem, the mu-opioid receptor (MOR-1) was stably expressed in murine neuroblastoma Neuro2A cells after transfection. The expressed receptor was negatively coupled to adenylyl cyclase through Gi/Go proteins, displayed high affinity ligand binding, and was expressed in high number (2.06 pmol/mg of [3H]diprenorphine binding sites). In addition, loss of ability of mu-opioids to acutely inhibit forskolin-stimulated cAMP formation was observed after 4-24 h of chronic exposure to these agonists with concentrations as low as 300-500 nM. The effects of chronic morphine or [D-Ala2,N-MePhe4,Gly-ol]enkephalin (DAMGO) administration were found to be time- and concentration-dependent. Cross 'tolerance' was also observed. Thus the IC50 value of DAMGO to inhibit adenylyl cyclase was increased by 27-fold from 4.3 nM in control cells to 117 nM in cells pretreated with 300 nM morphine; there was no effect on the inhibition of adenylyl cyclase mediated by muscarinic receptors. Further, receptor downregulation accompanied the desensitization process. However, different time-dependence for these two processes suggests, in line with other studies, that these are entirely different cellular adaptation processes. In addition, the opioid antagonist naloxone induced an acute increase in intracellular cAMP level (2-3 times above the control level) following chronic agonist exposure. This process was also concentration-dependent. Overall, these results suggest that the cell line utilized in this study has a homogeneous population of mu-opioid receptors, providing an ideal cellular model to study the molecular mechanisms underlying chronic morphine treatment.