An enkephalin derivative, [D-Ala2,Leu5]enkephalin N-[(2-nitro-4-azidophenyl)amino]ethylamide, has been synthesized as a photoaffinity label for the opiate receptor. This compound retains the full biological activity of [D-Ala2,Leu5]enkephalin in guinea pig ileum and mouse vas deferens tests with IC50 values of 4.4 and 2.6 nM, respectively, and inhibits the binding of [3H]naloxone to rat brain membrane preparation with an IC50 value of 2.5 nM. Photolysis of a muscle strip of the guinea pig ileum or of the mouse vas deferens in the presence of the peptide derivative caused irreversible inhibition of electrically stimulated contractions with high efficiencies (80 and 66%, respectively), while the inhibitory effect in the dark was fully reversed by washing. This irreversible inhibition during photolysis was completely prevented by the presence of [D-Ala2,Leu5]enkephalin. These results demonstrate that [D-Ala2,Leu5]-enkephalin N-[(2-nitro-4-azidophenyl)amino]ethylamide is a prominent candidate as a photoaffinity label for the opiate receptor.