The human NK1 tachykinin receptor in the astrocytoma cell line U 373 MG was characterized using selective agonists and antagonists described for this receptor in the rat. Specific [3H]substance P binding sites were present on cell homogenates, whereas [3H]neurokinin A or [3H]-senktide binding sites were absent. The binding was saturable and reversible. The binding of [3H]substance P was inhibited by very low concentrations of [L-Pro9]substance P and [Sar9,Met(O2)11]substance P; septide was approximately 1,000-fold less potent. The most potent peptide antagonist was trans-4-hydroxy-1-(1H-indol-3-ylcarbonyl)-L-prolyl-N-methyl-N-(phe nylmethyl)-L- tyrosineamide. The rank order of potency for the nonpeptide antagonists was (S,S)-CP 96,345 > (+/-)-CP 96,345 > (+/-)-2-chlorobenzylquinuclidinone > (R,R)-CP 96,345 > RP 67580 > RP 68651. In [3H]-inositol-labeled cells, substance P stimulated phosphatidylinositol turnover. A good correlation was found when the abilities of NK1 receptor agonists for stimulating inositol phosphate production and for inhibiting [3H]substance P binding were compared. Similarly, the binding and functional assays were well correlated for the antagonists. As a result of its high sensitivity and selectivity, the U 373 MG cell line thus appears an excellent tool for investigating the pharmacology of the human NK1 receptor.