The interaction of gentamicin with renal cortex of rats has been studied in vitro by means of a binding assay to brush border and basolateral membranes. Gentamicin specifically bound to plasma membrane fractions, compared to other subcellular fractions. Gentamicin binding to brush border and basolateral membranes was markedly inhibited by polycations such as spermine, and was slightly inhibited by high concentrations of tetraethylammonium. The treatment of phospholipase A2 to both types of membranes increased gentamicin binding, although the treatments by proteolytic enzymes and sulfhydryl reagent did not affect the binding. Gentamicin binding was increased in the brush border membranes treated with acidic phospholipids, whereas it was decreased in the membranes treated with calcium. Judging from the determination of membrane surface charge by metachromasy of cationic dye, basolateral membranes seemed to contain more anionic sites than brush border membranes. The alterations of gentamicin binding described above correlated with the changes of anionic charge on the membranes, indicating a charge interaction between gentamicin and anionic binding sites on the membranes. The addition of other aminoglycoside antibiotics to the incubation mixture induced significant reductions in the binding of gentamicin in the order of aminoglycosides according to their positive charge. The present results suggest that the characteristics of gentamicin binding to brush border and to basolateral membranes are essentially similar, and therefore the renal accumulation of gentamicin may be regulated by the transport of gentamicin across both plasma membranes.