Polyclonal antibodies were raised against isoxazolyl penicillins in rabbits after immunization with a cloxacillin-human serum albumin conjugate. The antisera were tested in direct and indirect competitive enzyme immunoassays (EIAs), using glucose oxidase or horseradish peroxidase conjugates of oxacillin, cloxacillin, or dicloxacillin, respectively, as the labelled antigen. The relative cross-reactivities of each test system with oxacillin, cloxacillin, and dicloxacillin, determined from the amount of antibiotic required for 50% inhibition of labelled antigen binding, were dependent on the antibiotic used as the labelled antigen. Other beta-lactam antibiotics did not cross-react in these test systems. In a direct EIA using a cloxacillin-horseradish peroxidase conjugate, cloxacillin and dicloxacillin in milk were detected at levels of 10 and 30 ng ml-1; the average recoveries at these levels were 102 and 84%, respectively.