A homogeneous substrate-labeled fluorescent immunoassay was developed for the measurement of kanamycin concentrations in serum. A fluorogenic drug reagent (FDR) (i-galactosyl-umbelliferone-tobramycin) was prepared that is nonfluorescent under the conditions of the assay but is hydrolyzed upon catalysis by l-galactosidase to yield a fluorescent product. Binding of the FDR to the antiserum to kanamycin prevented enzyme hydrolysis. The fixed level of FDR in the assay competed with kanamycin in the sample for a limited number of antibody-binding sites. Unbound FDR was hydrolyzed by ,3-galactosidase to release a fluorescent product that is proportional to the kanamycin concentration in the sample. The assay exhibited good sensitivity, precision, and accuracy and correlated well with other methods.Kanamycin is an aminoglycoside antibiotic which is effective in the treatment of severe infections caused by gramnegative bacteria (3). It has pharmacokinetic properties very similar to amikacin (4,8). It has been used to treat infections caused by Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., and Proteus spp., as well as other bacteria. Since kanamycin has a narrow therapeutic range, proper dosage administration is necessary to achieve maximum therapeutic efficacy and to avoid adverse effects, including nephrotoxicity and ototoxicity (5). Interpatient variability is also evident in response to a given dose of an aminoglycoside antibiotic. Therefore, therapeutic drug monitoring with a fast and accurate method is the most effective way of ensuring adequate therapy (6, 7, 10). We developed a homogeneous substrate-labeled fluorescent immunoassay (SLFIA) for the determination of total kanamycin levels in serum. The principle of the SLFIA, described previously (2,12,13), is based on the following findings. The kanamycin fluorogenic drug reagent (FDR), which is actually the cross-reacting compound ,-galactosyl-umbelliferone-tobramycin, is nonfluorescent under the conditions of the assay. (The FDR compound was used for convenience, since it had previously been synthesized for use in an SLFIA for the detection of tobramycin.) However, hydrolysis catalyzed by P-galactosidase (E. coli 3D-galactoside galactohydrolase; EC 3.2.1.23) yields a fluorescent product. When antiserum to kanamycin binds to the FDR, it is inactive as a substrate for ,-galactosidase, and fluorescence is inhibited. Kanamycin in the clinical sample competes with a constant amount of FDR for a limiting number of antibody-binding sites so that a fluorescent response is generated as a function of the kanamycin concentration.The performance of the kanamycin assay was evaluated for accuracy and precision, standard curve reproducibility, time dependence, sensitivity and cross-reactivity. Comparison of the assay with radioimmunoassays (RIA), highperformance liquid chromatography (HPLC), and gravimetric values is shown. Enzyme. 1-Galactosidase from E. coli (Sigma Chemical Co., St. Louis, Mo.) was assayed at 25°C in 50 mM Bicine (N,N-bis[2-hydroxyethyl]-glycine)...