The uptake of [3H]gentamicin by isolated rabbit renal proximal tubule brush border membrane vesicles was studied. Uptake was biphasic, with an initial rapid uptake followed by a prolonged slower phase. Approximately half of the total uptake represented binding; the other half represented transport into an intravesicular space. Scatchard analysis indicated the presence of two binding sites, differing in affinity (8 X 10(3) and 0.9 X 10(3) M-1) and number of sites per milligram of protein (1.2 and 3.7 nmol/mg of membrane protein, respectively). [3H]Gentamicin uptake was not affected by a Na+ electrochemical gradient, a valinomycin-generated (inside negative) K+ diffusion potential or the presence of phlorizin and D- of L-glucose. These findings indicate that the mechanism of uptake of the aminoglycoside was distinct from those of sugar and amino acids. Uptake of [3H]gentamicin was inhibited and reversed by the unlabeled aminoglycoside and by spermine. Spermine, on a molar basis, was as effective as gentamicin. These results suggest that gentamicin and spermine may have a common polyamine transport system and demonstrate the feasibility of further investigations to prevent aminoglycoside accumulation and possibly subsequent nephrotoxicity.