It is well known dermorphin is a potent and long-acting opioid peptide while its synthetic L-form is almost completely devoid of biological activity. We investigated whether the L-Ala2 residue might affect the affinity of the compound for opioid receptors or make [L-Ala2] dermorphin more sensitive to metabolic degradation. Dermorphin and [L-Ala2] dermorphin were assayed in [3H]naloxone binding to opioid receptors in rat brain preparations in the absence and presence of peptidase inhibitors bestatin, captopril and thiorphan. The synthetic [L-Ala2] dermorphin showed very low affinity for the opioid receptors. This was only slightly increased in the presence of the peptidase inhibitor bestatin, alone and in combination with captopril and thiorphan. The low affinity of [L-Ala2] dermorphin was not improved even when the binding assay was carried out at 0 degrees C. We suggest that the D-Ala2 residue is essential for the binding of dermorphin to the opioid receptors as well as for its pharmacological activity.