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BACKGROUND AND PURPOSE A 3H-labelled derivative of the novel small-molecule bradykinin (BK) B2 receptor antagonist JSM10292 was used to directly study its binding properties to human and animal B2 receptors in intact cells and to closely define its binding site. EXPERIMENTAL APPROACH Equilibrium binding, dissociation and competition studies with various B2 receptor ligands and [3H]-JSM10292 were performed at 4°C and 37°C. The experiments were carried out using HEK293 cells stably (over)expressing wild-type and mutant B2 receptors of human and animal origin. KEY RESULTS [3H]-JSM10292 bound to B2 receptors at 4°C and at 37°C with the same high affinity. Its dissociation strongly depended on the temperature and increased when unlabelled B2 receptor agonists or antagonists were added. [3H]-JSM10292 is cell membrane-permeant and thus also bound to intracellular, active B2 receptors, as indicated by the different 'nonspecific' binding in the presence of unlabelled JSM10292 or of membrane-impermeant BK. Equilibrium binding curves with [3H]-JSM10292 and competition experiments with unlabelled JSM10292 and [3H]-BK showed a different affinity profile for the wild-type B2 receptor in different species (man, cynomolgus, rabbit, mouse, rat, dog, pig, guinea pig). Characterization of B2 receptor mutants and species orthologues combined with homology modelling, using the CXCR4 as template, suggests that the binding site of JSM10292 is different from that of BK but overlaps with that of MEN16132, another small non-peptide B2 receptor ligand. CONCLUSIONS AND IMPLICATIONS [3H]-JSM10292 is a novel, cell membrane-permeant, high-affinity B2 receptor antagonist that allows direct in detail studies of active, surface and intracellularly located wild-type and mutant B2 receptors. [ABSTRACT FROM AUTHOR]