A series of deltorphin analogues was prepared by solid-phase peptide synthesis. Their opioid activity was evaluated in rat opiatic assay and their conformation was determined by two-dimension Nuclear Magnetic Resonance Spectroscopy. The analogues containing D-alanine acid at position 2 were much more potent in the assay than their corresponding isomers containing L-alanine acid at this position. The conformational analysis on NMR study in DMSO showed that C-terminal tetrapeptides of both deltorphin II and its L-alanine analog might form a 3(10) helix, which confirms that the substitution of D-amino acid at position 2 decreased the opioid activity.