AbstractGuanabenz (E‐2,6‐dichlorobenzylideneaminoguanidine acetate, Wy‐8678) and its identified metabolites were tested for their ability to displace [3H]clonidine from the cerebral α2‐receptors of rat and dog. The Kj's for inhibition of [3H]clonidine binding to rat membranes were 1.97, 0.96, 14.0, and 108 nM for clonidine, guanabenz, p‐hydroxyguanabenz, and the Z‐isomer of guanabenz, respectively. The other metabolites at concentrations of 10,000 nM did not displace [3H]clonidine. Scatchard analysis indicated single populations of binding sites with KD' s of 2.37 and 2.39 nM and Bmax's of 5.39 and 5.12 pmol/g for rat and dog preparations, respectively. Hill analysis yielded coefficients approximating unity, indicating a lack of cooperativity in binding. The high affinity of guanabenz relative to that of its metabolites for the α2‐receptor provided the basis for the development of an assay capable of discriminating guanabenz from its weakly hypotensive (p‐hydroxyguanabenz and the Z‐isomer of guanabenz) and pharmacologically inactive (2–6, dichlorobenzyl alcohol,2,6‐dichlorobenzaldehyde, 2,6‐dichlorobenzaldehyde azine, 2,6‐dichlorobenzaldehyde semicarbizone, creatinine adduct of 2,6‐dichlorobenzaldehyde, and an isomer of the creatinine adduct) metabolites. Antihypertensive agents with different mechanisms of action such as indoramin, prazosin, debrisoquine, and bethanidine bound little, if at all, and thus will not interfer