The disposition of the potent antihypertensive drug clonidine has been poorly understood through the lack of a convenient and sensitive assay. A radioimmunoassay for clonidine has been developed and is capable of detecting as little as 10 pg of clonidine. 2,6-Dichlorophenyl-guanidine, a known metabolite of clonidine, did not cross-react with the antiserum whereas another metabolite, 4-hydroxyclonidine, was as potent as clonidine in displacing labeled clonidine from the antibody. However, a simple solvent extraction step before the radioimmunoassay selectively extracted clonidine from a mixture of clonidine and 4-hydroxyclonidine in alkaline plasma and this procedure permitted a specific assay for clonidine. The plasma levels of clonidine in rats after the administration of a hypotensive dose (100 microgram/kg i.v.) were determined by radioimmunoassay and these data indicated that the disposition of clonidine conforms to an open two-compartment, pharmacokinetic model. Clonidine rapidly accumulated in the brain as shown by the attainment of peak concentrations within 2 min of i.v. injection.