Side chain modifications were introduced to endomorphin 2 (E2) to improve its binding properties and biological activity. A number of C-terminal modifications decreased the binding affinity to the mu-opioid receptor and the intrinsic activity in rat brain membranes. The exception was E2-ol, which showed increased binding affinity to MOR and higher potency in stimulating [S-35]GTPgammaS binding. N-methylation of Phe(3) (MePhe(3)) attenuated the binding affinity and produced a rightward shift of [S-35]GTPgammaS binding curves. All derivatives had lower intrinsic activity than E2. Some of the modified peptides partially inhibited, while YPF-benzyl-allyl-amide fully inhibited, the E2 or [D-Ala(2),MePhe(4),Gly(5)ol]enkephalin stimulated [S-35]GTPgammaS binding. Marked differences were found between the results obtained using tritiated E2, tritiated naloxone, and [S-35]GTPgammaS binding, indicating the possible involvement of multiple binding sites. The data presented demonstrate that the C-terminal amide group has an essential role in the regulation of the binding and the agonist/antagonist properties of E2. (C) 2002 Elsevier Science.