The mechanism(s) by which systemically-administered aminoglycosides enter the cochlea remain poorly understood. To elucidate which mechanisms may be involved, we co-administered different molar ratios of gentamicin and fluorescent gentamicin (GTTR) to mice in three different regimens: (1) gentamicin (150, 300 or 600mg/kg) containing a constant 300:1 molar ratio of gentamicin:GTTR; (2) 300mg/kg gentamicin containing a variable molar ratio of gentamicin:GTTR (150:1-600:1), or (3) an increasing dose of gentamicin (150-900mg/kg), each dose containing 1.7mg/kg GTTR. Three hours later, cochleae were fixed and examined by confocal microscopy. First, increasing doses of a constant molar ratio of gentamicin:GTTR, resulted in increasing intensities of GTTR fluorescence in hair cells and strial tissues. Second, a fixed gentamicin dose with increasing molar dilution of GTTR led to decreasing GTTR fluorescence in hair cells and strial tissues. Third, a fixed GTTR dose with increasing molar dilution by gentamicin led to decreased GTTR uptake in hair cells and marginal cells, but not intra-strial tissues and capillaries. Thus, only hair cell and marginal cell uptake of GTTR is competitively inhibited by gentamicin, suggesting that a regulatable barrier for gentamicin entry into endolymph exists at the interface between marginal cells, the intra-strial space and intermediate cells.