Fluorescence polarization immunoassays of the aminoglycoside antibiotics gentamicin, tobramycin, and amikacin in plasma and serum are described and shown to be clinically useful. The aminoglycoside tracers were prepared by reacting the parent compounds with 5-[(4,6-dichlorotriazin-2-yl)-amino] fluorescein. Antisera specific for the compounds were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum are combined and, after a 15-min incubation at ambient temperature, the polarization of the fluorescence of the tracer is determined in a specially designed fluorometer. The assays are designed to give accurate trough (i.e., minimum during therapy) values and to be free of matrix effects. Severely icteric samples may interfere, but this can be overcome by blank subtraction. The performance of the assays with clinical specimens compared favorably with that of some commercially available assays.