We examined the effect of simultaneously incorporating proline or proline-amino acid chimeras in positions 9, 10, and/or 11 of substance P, on the affinity for the two NK-1 binding sites and on second-messenger activation. Because these 3-substituted prolines constrain not only the (phi,psi) values of the peptide backbone, but also the chi space of the amino acid side chain, we were able to gather data on the structural requirements for high-affinity binding to the NK-1 receptor. We were able to confirm that this C-terminal component is crucial and that it should adopt an extended conformation close to a polyproline II structure when bound to the receptor. The partial additivity of these constraints, more specifically, for the NK-1M site, suggests that the peptide backbone flexibility around the hinge-point residue Gly9 is essential to subtly position crucial side chains.