A series of [Trp4]enkephalin analogues designed to prevent the formation of various intramolecular hydrogen bonds was synthesized by the Merrifield method. In vitro opiate activities of [Trp(NαMe)4,Met5]enkephalin, [Trp4,Leu(NαMe)5]enkephalin, [Trp4,Pro5]enkephalin and [Tyr(OMe)1,Trp4,Met5]enkephalin and of their corresponding [Phe4]analogues were determined with the rat brain receptor assay and the guinea pig ileum assay. The discrepancies between potencies observed in the two assay systems provided further evidence for the existence of several subclasses of opiate receptors. The significant activity obtained with the Nα-methylated analogues indicates that hydrogen bonds involving the backbone amino groups in positions 4 or 5 are not required in the biologically active conformation. A conformational study by fluorescence techniques was performed under monomeric conditions in dilute aqueous solution (10-5 M). Average intramolecular Tyr-Trp distances were determined by evaluation of singlet-singlet resonance energy transfer on the basis of the Forster equation. Distances ranged from 8.8 to 10.7 Å and are thus comparable to the Tyr-Trp distance observed in [Trp4,Met5]enkephalin (9.3 Å). These findings indicate that folded conformations of enkephalin in H2O may be brought about by solvent interactions and need not be stabilized by intramolecular 4→1 or 5→2 hydrogen bonds between amino and carbonyl groups of the peptide backbone or between the tyrosine hydroxyl and a backbone carbonyl group. The results are discussed in relation to various proposed solution conformations of enkephalin.