Methionine enkephalin release was evoked by depolarization of slices from rat striatum with potassium. In the presence of 0.1 microM thiorphan [(N(R,S)-3-mercapto-2-benzylpropionyl)glycine], a potent inhibitor of enkephalin dipeptidyl carboxypeptidase (enkephalinase), the recovery of the pentapeptide in the incubation medium was increased by about 100 percent. A similar effect was observed with the dipeptide phenylalanylalanine, a selective although less potent enkephalinase inhibitor. Inhibition of other known enkephalin-hydrolyzing enzymes--aminopeptidase by 0.1 mM puromycin or angiotensin-converting enzyme by 1 microM captopril--did not significantly enhance the recovery of released methionine enkephalin. These data indicate that enkephalinase is critically involved in the inactivation of the endogenous opioid peptide released from striatal neurons.