Four cell lines producing monoclonal antibodies were obtained by fusion of NS1 myeloma cells with splenocytes of BALB/C mice immunized with only 1 microgram of each staphylococcal enterotoxin A, B, C1 and D by a modified technique of intrasplenic boosting. This procedure was considerably more efficient than the more commonly used intravenous boosting. The antibodies EC-A1, EC-B1, EC-C1 and EC-D1, all of the IgG1 subclass, have high affinities for the corresponding enterotoxins A, B, C1 and D, with dissociation constants of 1.4, 2.8, 1.4 and 1.5 nM respectively; in addition EC-B1 showed a high affinity (2.1 nM) for enterotoxin C1. All these antibodies recognize, by immunoblotting, the homologous purified enterotoxins as well as enterotoxins from the bacterial culture supernatants. A rapid indirect double sandwich ELISA using a pair of antibody preparations was developed, where monospecific monoclonal antibodies were used to coat plastic plates and polyspecific rabbit antibodies were used to detect the enterotoxins under field conditions. These antibodies which are capable of immunoadsorbing the enterotoxins from staphylococcal culture filtrates and from natural fluids such as milk, were used to immunopurify enterotoxins A, C1 and D. The homogeneity and integrity of the affinity purified toxins A, C1 and D was verified by direct automated Edman degradation and yielded single amino terminal sequences which were moderately homologous to those published previously for B and C1 enterotoxins.