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[3H]gentamicin and [14C]gentamicin samples were purified by Sephadex column chromatography and separated by an HPLC technique into the three major, medicinally active gentamicin components. These separated components were used in equilibrium dialysis studies to determine their percent binding to serum proteins. The bindings of the components were inversely related to concentrations of ionized calcium and magnesium. When dialyzed against a buffer containing physiological concentrations of the divalent cations, the binding of the C(1) component was 2.2 +/- 1.0%, the binding of the C(1a) component was 1.2 +/- 1.9%, and the binding of the C(2) component was 5.0 +/- 2.0%. The percent bindings are not identical and, due to their low values, probably have negligible clinical significance. The radioactive composition and purity of the 3H- and 14C-labeled gentamicin samples differed and these may be important factors in the variance of reported gentamicin bindings.