Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.