Analogues of leucine-enkephalinamide containing N-terminal cis- or trans-4-(3-hydroxyphenyl)-4-methylpipecolic acid were prepared to examine the conformational requirements of the N-terminal tyrosyl residue in opioid activity. The diastereomeric amino acids were prepared and purified by semipreparative HPLC before incorporation into the peptide. Spectroscopic analysis based on proton nuclear Overhauser enhanced differential spectroscopy (NOEDS) allowed assignment of the cis and trans stereochemistry. Despite spatial analogy between the trans isomer 5 and leucine-enkephalinamide, it possessed neither opioid agonist nor antagonist activity in the guinea pig ileal longitudinal muscle (GPI) or mouse vas deferens (MVD) preparations. Possible explanations for this inactivity are discussed.