We have characterized the binding of a novel radioligand, [3H] FK888, to neurokinin (NK)1 receptors in guinea pig lung membranes and localized its binding in guinea pig lung sections by autoradiography. Lung membranes were incubated with [3H] FK888 at 25 degrees and the assays were terminated by rapid filtration; nonspecific binding was defined as binding in the presence of 1 microM concentrations of the nonpeptide NK1-selective antagonist CP-96,345. Kinetic analysis showed that specific binding of [3H] FK888 (approximately 70% of total binding) was rapid, reaching a plateau by 20 min, and that binding was reversed by addition of 1 microM CP-96,345, giving a kinetic Kd of 0.46 nM. Binding of [3H] FK888 was saturable at approximately 1 nM, and equilibrium binding analysis gave a Kd of 0.32 +/- 0.03 nM and a Bmax of 46.9 +/- 7.1 fmol/mg of protein (four experiments). In competition studies, substance P, CP-96,345, and FK888 competed for [3H] FK888 binding, but NKA, NKB, and NK2-selective antagonists such as SR48968 and L-659,877 did not. Guanosine-5'-O-(3-thio)triphosphate significantly shifted the competition curve for substance P competition against [3H]FK888 binding to a lower affinity state, confirming that NK1 receptors are coupled to a G protein. Autoradiographic mapping in cryostat sections of lung showed that [3H]FK888 binding was dense over smooth muscle of all airways, with moderate binding over epithelium of bronchi and bronchioles as well as submucosal glands of trachea. No significant labeling of blood vessels was observed. [3H]FK888 binds to NK1 receptors in guinea pig lung and may be a useful tool for studying the expression and regulation of NK1 receptors.