A dipstick dot-ELISA for the detection of gentamicin in milk of dairy cattle is reported for the first time. The test is based on a sandwich ELISA using high affinity monoclonal antibodies to gentamicin. Antibodies were adsorbed to nitrocellulose filters, blocked, dried, and stored for several weeks before use. The dipstick ELISA detected gentamicin at a concentration of 0.1 microgram/ml and produced strongly positive results at 0.2 microgram-0.3 microgram/ml. This ELISA is highly specific and no false positives were detected when tested against various aminoglycoside analogs including streptomycin, kanamycin, bekanamycin, amikacin, neomycin, and tobramycin. Further, the elimination in cow milk of gentamicin residues following intramammary administration of the drug was studied in two dairy cattle using dot-ELISA. Milk gentamicin levels were detected at post injection hours up to 120 hr in each of the two dairy cattle. It therefore, appears that gentamicin residues can still be detected in milk after 5 days using dot-ELISA. Based on the simplicity of performance and the economical nature of the test system, dipstick is recommended as a suitable method for wide scale use in field studies and diagnostic laboratories.