Polyclonal antisera against streptomycin were prepared by using a streptomycin‐oxime derivative coupled to bovine serum albumin for the immunization of rabbits. The specificity and sensitivity of these antibodies were tested in a competitive assay using a streptomycinenzyme conjugate (prepared by coupling a streptomycin‐hydrazone derivative to horseradish peroxidase) in a double antibody solid phase technique. The only detectable cross‐reactivity of the assay system with other aminoglycoside antibiotics and other substances similar in structure was shown to be with dihydrostreptomycin of about 148.7%. The detection limit in buffer solution was 0.6 ng ml‐1for streptomycin and 0–4 ng ml‐1for dihydrostreptomycin. Employing rapid sample preparation procedures, streptomycin and dihydrostreptomycin were detected in milk at levels as low as 6 and 0.8 ng ml‐1respectively.