A specific, sensitive, and simple radioimmunoassay was developed for the oral hypoglycemic drug glibenclamide, N-4-[2-(5-chloro-2-methoxy-benzamide)- ethyl]-benzenesulfonyl-N'-cyclohexylurea. Antiserum against glibenclamide was obtained from rabbits immunized with an antigen prepared by conjugating the diazonium salt of N-(p-amino-benzamidoethyl)- benzenesulfonyl-N'-cyclohexylurea to bovine serum albumin through the diazocoupling. [3H]glibenclamide was used as a tracer. Dextran-coated charcoal was used to separate bound and free [3H]glibenclamide in the reaction mixture. The radioimmunoassay is able to determine as little as 25 pg of glibenclamide directly in plasma without the need for extraction. The antiserum used for the assay was highly specific for glibenclamide, and did not cross-react with two known major metabolites of glibenclamide. Comparable values of glibenclamide in dog plasma were obtained by radioimmunoassay and liquid chromatography. Plasma concentrations of glibenclamide in diabetic patients on glibenclamide treatment can be determined by radioimmunoassay, and the method has been applied to the routine assay of clinical samples. This radioimmunoassay seems to be useful for monitoring plasma glibenclamide concentrations.