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A rapid, specific method for measuring gentamicin in plasma by high-pressure liquid chromatography was developed. After deproteinization, gentamicin in the supernate was dansylated and extracted into ethyl acetate. The organic extract was chromatographed on a microparticulate reversed-phase column, which was eluted with aqueous acetonitrile. Use of the dansyl derivative enables fluorometry, for more sensitive quantitation. Various factors that could affect the assay sensitivity were investigated. With 0.2-ml plasma samples, the method can accurately measure as little as 1 mg of gentamicin per liter. We encountered no interferences from plasma supplemented with various drugs or plasma of patients who were on therapy with other drugs. This method can also separate gentamicin C1 from C1a and C2, all of which are present in various ratios in commercial dosage forms. This method is also applicable to gentamicin determination in urine.