An immunoassay was developed for the detection of sulfamethoxazole reactive IgE antibodies in the sera of patients who experienced life threatening anaphylactic reactions following the ingestion of co-trimoxazole (trimethoprim and sulfamethoxazole). Patients who had significant levels of sulfamethoxazole reactive IgE antibodies in their sera did not have IgE antibodies that reacted with trimethoprim-Sepharose. Inhibition experiments with a number of sulfonamides to determine the fine structural specificities of the sulfamethoxazole reactive IgE antibodies in three patients revealed that sulfamethoxazole and, depending on the serum, sulfamerazine and sulfamethizole, were the most potent inhibitors of IgE binding, whereas the parent sulfonamide, sulfanilamide, was a very poor inhibitor. From a detailed examination of structure-activity relationships, we concluded that the 5-methyl-3-isoxazolyl group on the sulfamethoxazole molecule was the allergenic determinant for all three patients with the 5-methyl group being particularly important for IgE antibody recognition. The assays for the detection of IgE antibodies to sulfamethoxazole and trimethoprim should prove useful for the diagnosis of immediate hypersensitivity to co-trimoxazole and perhaps for monitoring drug therapy in AIDS patients where a high incidence of adverse reactions to co-trimoxazole has been reported.