Our previous studies showed that coadministration of cytochrome c and a 20-residue basic peptide, N-WASP181–200 (NISHTKEKKKGKAKKKRLTK, pI=10.87) inhibits renal accumulation of gentamicin. In this study, we examined effects of ligands of megalin, an endocytic receptor involved in renal uptake of gentamicin, and basic peptides including N-WASP180–200 and its mutant peptides on gentamicin binding to isolated rat renal brush-border membrane (BBM). Gentamicin binding to BBM was inhibited by megalin ligands, basic peptide fragments of cytochrome c, and N-WASP181–200 in a concentration-dependent manner. Klotz plot analysis showed that N-WASP181–200 inhibited the binding of gentamicin in a competitive manner. By substituting glycines for lysines in N-WASP181–200 at positions 9 and 15, the inhibitory effect on gentamicin binding to BBM was reduced, which may be related to a decrease in the α-helix content in the peptide. Gentamicin binding to BBM treated with trypsin, in which megalin completely disappeared, was significantly but not completely decreased compared with the native BBM. In addition, treatment of BBM with trypsin led to a decrease in the inhibitory effect of N-WASP181–200 on gentamicin binding. These observations support that megalin ligands and basic peptides including N-WASP181–200 decrease renal accumulation of gentamicin by inhibiting its binding to BBM of proximal tubule cells, partly interacting with megalin. In addition, the α-helix conformation may play an important role in the inhibitory effect of N-WASP181-200 on the binding of gentamicin to BBM.