This article describes new deltorphin I analogs in which phenylalanine residues were replaced by the corresponding (R) or (S)-α-benzyl-β-azidoalanine, α-benzyl-β-(1-pyrrolidinyl)alanine, α-benzyl-β-(1-piperidinyl)alanine, and α-benzyl-β-(4-morpholinyl)-alanine residues. The potency and selectivity of the new analogs were evaluated by a competitive receptor binding assay in the rat brain using [3H]DAMGO (a μ ligand) and [3H]DELT (a δ ligand). The affinity of analogs containing (R) or (S)-α-benzyl-β-azidoalanine in position 3 to δ-receptors strongly depended on the chirality of the α,α-disubstituted residue. The conformational behavior of peptides modified with (R) or (S)-α-benzyl-β-(1-piperidinyl)Ala, which displays the opposite selectivity, was analyzed by 1H and 13C NMR. The μ-selective Tyr-d-Ala-(R)-α-benzyl-β-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2 lacks the helical conformation observed in the δ-selective Tyr-d-Ala-(S)-α-benzyl-β-(1-piperidinyl)Ala-Asp-Val-Val-Gly-NH2. Our results support the proposal that differences between δ- and μ-selective opioid peptides are attributable to the presence or absence of a spatial overlap between the N-terminal message domain and the C-terminal address domain.