The conformation of substance P (free acid) (SPOH) has been investigated in dimethylsulfoxide (DMSO), water and dipalmitoylphosphotidylcholine (DPPC) bilayers by two-dimensional NMR and restraint molecular dynamics simulations. The observed NOE patterns for SPOH in these media are very much different from each other. Molecular modeling of the conformation of SPOH by incorporating NOEs as distance restraints shows wide differences in its conformation in three media. The main structural features for SPOH in DMSO are γ-bends at Pro⁴ and Phe⁷ along with a non-specific bend around Lys³-Pro⁴-Gln⁵-Gln⁶, which are stabilized by Lys³CO→Gln⁵NH, Gln⁶CO→Phe⁸NH hydrogen bonding. The more flexible conformation of SPOH in water is transformed to an ordered structure after incorporation in DPPC bilayers. The conformation of SPOH in DPPC bilayers is characterized by γ-bends at Pro⁴, Gln⁶ and Phe⁷, which are stabilized by hydrogen bonding between Lys³CO→Gln⁵NH, Gln⁵CO→Phe⁷NH and Gln⁶CO→Phe⁸NH, respectively. The absence of biological activity in SPOH has been attributed to the absence of any helix like structure at the central residues and absence of any interresidue interaction with C-terminal OH group, in DPPC bilayers, a feature shown to be an important prerequisite for SP and SP agonists to bind to the NK1 tachykinin receptor.