Polyclonal antibodies against sulfamethazine, sulfadiazine, sulfamethoxypyridazine and trimethoprim were prepared by using bovine serum albumin conjugates of the respective substances as antigens for the immunization of rabbits. The specificity and sensitivity of these antibodies were tested by using the respective drug coupled to horseradish peroxidase as the labelled antigen in a competitive assay. The antiserum against sulfamethazine showed relative cross‐reactivities of 100 and 56% with sulfamethazine and sulfamerazine, respectively. The antiserum against sulfadiazine showed relative cross‐reactivities of 100, 12, 11 and 10% with sulfadiazine, sulfasalazine, sulfamerazine and sulfathiazole, respectively. The assays for sulfamethoxypyridazine and trimethoprim showed no relative cross‐reactivity above 1%. The detection limits (in buffer solution) for sulfadiazine, sulfamethazine, sulfamethoxypyridazine and trimethoprim were at 1.1, 1.3, 2.8 and 6.0 ng/ml, respectively. The recovery of the sulfonamides from milk samples, artificially contaminated at levels of 5, 10, 50 and 100 ppb, was between 68 and 99%. Recovery of trimethoprim from artificially contaminated milk samples was about 90% at concentrations of 50 and 100 ppb.