The aim of this study was to develop and evaluate an enzyme immunoassay (EIA) for the cephalosporin antibiotic in milk, in combination with a new microbiological test system (brilliant black reduction test, BRT-P). Polyclonal antibodies against cefquinome were produced in rabbits, using cefquinome-keyhole limpet hemocyanine as the immunogen. These antibodies and a cefquinome-glucose oxidase conjugate were used in a competitive indirect EIA. The detection limit for cefquinome in milk was 1.5 ng ml(-1), recoveries were 80-128% at 4-40 ng ml(-1). Cross-reactivities with other cephalosporins/penicillins were all <1%. The EIA was used to determine cefquinome in incurred raw milk, the BRT-P (detection limit ≈ 20 ng ml(-1)) and a receptor assay (ßeta-s.t.a.r., detection limit ≈ 15 ng ml(-1)) were used in parallel. Five lactating cows, suffering from clinical mastitis, were treated with cefquinome by simultaneous intramammary and intramuscular injection. Cefquinome residues (maximum 10-27 μg ml(-1)) were most exclusively found in the udder quarter which was treated intramammary, residue levels in the other three quarters were low (<20 ng ml(-1)). Even in milk from intramammary-dosed quarters, residue levels fell below European Union maximum residue level (MRL, 20 μg kg(-1)) 2 days before the end of the withdrawal period. EIA, BRT-P, and ßeta-s.t.a.r. results showed acceptable agreement for milk samples, but the newly developed EIA is superior in aspects of sensitivity. In conclusion, this is the first one description of immunoassay and microbiological tests capable to determine cefquinome in milk at the MRL in incurred sample material.