Pharmacological and biochemical assays were performed to characterize SR 142801, a new NK-3 receptor antagonist, and its [R]-enantiomer, SR 142806. The compounds were tested (1) in the guinea pig isolated ileum stimulated with [MePhe7]NKB (NK-3 system) in order to evaluate onset and duration of action and to estimate the apparent affinity of the antagonist in terms of pA2 at 140 min after application; (2) in 6 selected monoreceptor systems, the rabbit (rb) vena cava for the NK-1rb receptor, the rabbit pulmonary artery and the hamster (hs) urinary bladder for the NK-2rb and NK-2hs receptors, the rat (r) portal vein for the NK-3r receptor, and in two multireceptor systems adequately treated with NK-1 or NK-2 receptor antagonists to obtain monoreceptor-mediated biological responses (the rat urinary bladder treated with SR 48968 for evaluating the NK-1r and the guinea pig-gp-ileum treated with CP-99994 for measuring the antagonist affinity on the NK-3gp receptor), in order to evaluate the antagonist selectivity, and (3) in various plasma membrane preparations containing NK-3-binding sites from rats, guinea pigs, and man. The data presented indicate that SR 142801 is a potent, fairly selective non-peptide antagonist of the functional (pA2 9.4) and binding (Ki 0.11 nmol/l) site of the guinea pig and human (Ki 0.21 nmol/l) NK-3 receptors, while being much less active on the Nk-3 receptors of other species, particularly the rat (pA2 7.0; Ki 15 nmol/l). SR 142801 shows a slow onset of action and acts as a long-lasting irreversible antagonist, specific for neurokinin receptors, especially the NK-3 sites of guinea pigs and man.