We investigated the receptor-binding properties and potencies of FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-prolyl]-N- phenylmethyl-3-(2-naphthyl)-L-alaninamide), a tachykinin receptor antagonist, for the rat and human tachykinin receptor subtypes (NK1, NK2 and NK3) expressed in transfected mammalian cells. In displacement analyses, using membrane preparations derived from monkey kidney COS-7 cells transiently expressing tachykinin receptor subtypes, FK888 showed a subtype selectivity for NK1 receptor and its affinity for the human NK1 receptor was 320-fold higher than that for the rat NK1 receptor, demonstrating species difference in its binding affinity. This was in marked contrast to FK224 (N-[N2-[N-[N-[N-[2,3-didehydro-N-methyl-N-[N-[3-(2-pentylphenyl )- propionyl]-L-threonyl]tyrosyl-L-leucynyl]-D-phenylalanyl]-L- allothreonyl]-L-asparaginyl]-L-serine-n-lactone) that was selective for NK1 and NK2 receptors with similar affinities for the rat and human receptors. In Chinese hamster ovary cells permanently expressing the human NK1 receptor, FK888 inhibited the substance P-induced phosphatidylinositol hydrolysis and produced a parallel shift in the dose-response curve for substance P. Schild analysis of the antagonism of phosphatidylinositol hydrolysis by FK888 yielded a pA2 value of 8.9 and a slope of 0.97 of the regression line. FK888 itself showed no stimulatory effect on phosphatidylinositol hydrolysis in Chinese hamster ovary cells expressing the human NK1 receptor. Thus, FK888 is a potent, competitive and selective antagonist for human NK1 receptor.