A radioligand binding assay for the HERG (human ether-a-go-go-related gene) K(+) channel was developed to identify compounds which may have inhibitory activity and potential cardiotoxicity. Pharmacological characterization of the [(3)H]astemizole binding assay for HERG K(+) channels was performed using HERG-expressing HEK293 cells. The assay conditions employed yielded 90% specific binding using 10 microg/well of membrane protein with 1.5 nM of [(3)H]astemizole at 25 degrees C. The K(d) and B(max) values were 5.91 +/- 0.81 nM and 6.36 +/- 0.26 pmol/mg, respectively. The intraassay and interassay variations were 11.4% and 14.9%, respectively. Binding affinities for 32 reference compounds (including dofetilide, cisapride, and terfenadine) with diverse structures demonstrated a similar potency rank order for HERG inhibition to that reported in the literature. Moreover, the [(3)H]astemizole binding data demonstrated a rank order of affinity that was highly correlated to that of inhibitory potency in the electrophysiological studies for HERG in HEK293 (r(SP) = 0.91, P<0.05). In conclusion, the [(3)H]astemizole binding assay is rapid and capable of detecting HERG inhibitors.