The three mammalian tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), exert their physiological effects through specific receptors, NK1, NK2 and NK3, respectively. However, homologous binding studies have recently demonstrated that, contrary to the generally accepted belief, NKA could bind NK1 receptor with high affinity (Hastrup and Schwartz, 1996). Using COS-7 cells expressing the human NK1 receptor, we show that two simultaneous point mutations (E193L and V195R) in a restricted five amino acid sequence (the (193-197) region), selected because of its hydropathic complementarity with the common C-terminal extremity of tachykinins, abolish both the high-affinity binding and highly potent biological activity of NKA, without affecting those of SP. In addition, the same mutations also suppressed the high functional activity of septide, a synthetic SP atypical agonist ([pGlu6-Pro9] SP 6-11). These results suggest that the (193-197) region, located at the end of the second extracellular loop of the receptor, could be part of a common high-affinity binding domain for both NKA and septide, distinct from the SP binding site.