We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. (R)-N-[2-[Acetyl[3H3][(2-methoxyphenyl)-methyl]amino]- 1-(1H-indol-3-ylmethyl) ethyl][1,4'-bipiperidine]- 1'-acetamide ([3H]LY303870) binds to a single class of sites with an equilibrium KD of 0.22 nM and a Bmax of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC50 of 0.56 nM, whereas the less active (S)-enantiomer (LY306155) was substantially less potent. The nonpeptide NK-1 antagonists (+/-)-CP96,345 and (+/-)-RP 67580 had IC50 values of 0.74 and 49 nM, respectively. Substance P (SP) was also a potent inhibitor with with an IC50 of 3.1 nM. The inhibition by SP could be separated into two components: a high-affinity component with a Ki of 0.53 nM and a lower-affinity component with a Ki of 155 nM. Addition of 100 microM guanylyl 5'-imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low-affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [3H]-LY303870 was not changed by the presence of 100 microM Gpp(NH)p. The distribution of [3H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK-1 receptors labeled by [3H]SP. Therefore, [3H]LY303870 is a potent and selective antagonist radioligand for NK-1 receptors in guinea pig brain. In addition, regulation of NK-1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors.