Radionuclides currently used in clinical medicine were evaluated for their possible interference with radioenzymatic assays of aminoglycoside antibiotics. Of the radiopharmaceuticals tested, only (67)Ga citrate interfered with the radioenzymatic assay of gentamicin. Radioenzymatic assay of serum samples obtained from patients receiving (67)Ga yielded gentamicin concentrations falsely elevated by more than 1 mug/ml for approximately 1 week after (67)Ga administration. A procedure was developed to eliminate (67)Ga interference with radioenzymatic assays of aminoglycoside antibiotics. After (67)Ga citrate was spotted onto phosphocellulose filter disks, the filters were immersed in 7.2 N HCl, and radioactivity was removed by successive extractions of the acid phase with diisopropyl ether. After three extractions, less than 0.1% of the original radioactivity remained. Similar extraction of disks containing (14)C-adenylylated gentamicin or tobramycin or (14)C-acetylated amikacin had no effect on (14)C radioactivity. The concentrations of aminoglycosides in serum standards supplemented with (67)Ga citrate were determined accurately by radioenzymatic assays followed by extraction with diisopropyl ether. Concentrations of gentamicin in six serum samples from patients injected with (67)Ga during gentamicin therapy, as determined by radioenzymatic assay and extraction, were within 9% of the results obtained by reassay of the same samples after the decay of (67)Ga.