Prosthesis of non-critical parts of a polypeptide backbone is an attractive strategy to simplify bioactive peptides. This approach was applied to an opioid neuropeptide, Met-enkephalin, in which two adjacent Gly2-Gly3 residues were replaced with a series of non-peptidic backbone spacers varying in length and/or physicochemical properties. The backbone spacers did not affect the overall structural properties of the analogues, but they did dramatically reduce their affinities and agonist activities toward delta- and mu-opioid receptors. Molecular modeling suggested that the decrease of the affinity of Met-enkephalin to delta-opioid receptor could be accounted for by the loss of a single hydrogen bond. Remarkably, the analogues containing the most isostere spacers retained potent antinociceptive and anticonvulsant properties that were comparable to that of the endogenous peptide. This unexpected high in vivo potency could not be accounted for by an increase in metabolic stability. Moreover, the antiepileptic activity could not be reversed by opioid receptor antagonists. In summary, the results obtained with the analogues containing backbone spacers suggest a novel mechanism for seizure control in the brain that involves alternative non-opioid signaling.