WIN 64821, a secondary metabolite produced by Aspergillus sp. (ATCC 74177) was found to inhibit radiolabeled substance P (SP) binding in a variety of tissues, including those of human origin. This compound inhibited, in a competitive manner, the binding of SP with Ki values ranging from 0.24 microM in human astrocytoma U-373 MG cells to 7.89 microM in rat submaxillary membranes. Additionally, WIN 64821 was found to inhibit 125I-NKA binding to the NK2 receptor in human tissue at a concentration equivalent to its NK1 activity (0.26 microM). The inhibitory activity of WIN 64821 against an NK3 selective ligand, 3H-senktide, was found to be much weaker (Ki = 15.2 microM). WIN 64821 was also evaluated in NK1 functional assays and was found to be a competitive antagonist of SP-induced contractility in the guinea pig ileum (pA2 = 6.6) as well as an inhibitor of SP-induced 45Ca2+ efflux from human astrocytoma U-373 MG cells (IC50 = 0.6 microM). In a rat vas deferens model, WIN 64821 inhibited eledoisin-induced contractility with an IC50 of 3.4 microM indicating functional antagonism at the NK2 receptor. The data presented in this study provide biochemical, pharmacological and functional evidence supporting WIN 64821 as a competitive neurokinin antagonist.