This report describes a sensitive and specific radio-immunological method for determining serum levels of the oral anti-diabetic agent 1-(p-[2-(5-chloro-2-methoxy-benzamido)-ethyl]-benzensulfonyl)-3-cyclohexylurea (glibenclamide) and its metabolites. The antigen was prepared by coupling a metabolite to bovine serum albumin. Antibodies could be demonstrated in serum after immunisation of rabbits. The separation of free and antibody-bound glibenclamide was achieved by a dextran-charcoal suspension. Presence of serum did not influence the binding characteristics. The limit of detection was 20 ng/ml. The affinity of the metabolites differed only slightly from that of glibenclamide. The presence of related drugs from the sulfonylurea series such as tolbutamide, glibornuride and the sulfonamide sulfamethoxazol did not affect the determination. Only closely related substances showed a variable degree of affinity towards antibodies. As an example of the possible application of this method, the serum concentration of glibenclamide was determined over a period of 8 h after a single i.v. injection to a volunteer. The data are in close accordance with the results of authors using radioactive glibenclamide.