The present study demonstrates a conditional, agonist-dependent phosphorylation of the mu-opioid receptor (MOR-1) by cyclic AMP-dependent protein kinase (PKA) in membrane preparations of MOR-1-transfected neuroblastoma Neuro2A cells. Opioid agonist-dependent phosphorylation occurs in a time- and concentration-dependent manner (EC50 approximately 40 nM) and can be abolished by the receptor antagonist naloxone. Stoichiometric analysis indicates incorporation of a maximum of 6 mol of phosphate/mol of receptor in the presence of 1 microM morphine and 6 nM PKA. Although morphine and related alkaloids as well as some peptide agonists (PLO17 and beta-endorphin) stimulated phosphorylation of MOR-1 by PKA, the potent mu-opioid-selective peptide [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) or other enkephalin analogues such as [D-Ala2]-Met5-enkephalinamide (DALA), [D-Ala2,D-Leu5]-enkephalin (DADLE), and Met5-enkephalin had no effect. The lack of the effect of DAMGO on MOR-1 phosphorylation state was evident also after chronic pretreatment. These results suggest the existence of different agonist-dependent conformations of MOR-1. Furthermore, phosphorylation may be a useful parameter with which to identify different agonist-receptor conformations.