The characteristics of aminoglycoside uptake in the inner ear were investigated in the isolated crista ampullaris. The organ was incubated with radiolabeled gentamicin, and the stable, nonexchangeable radioactivity was considered the intracellular pool of the drug. Gentamicin was transported against a concentration gradient, and the resulting tissue to medium ratios ranged from 1.4 to 18.6. Transport was inhibited by reduction in temperature or by the addition of metabolic blockers, indicative of an energy-dependent component. The uptake system showed at least two sites, the first a high-affinity site with a dissociation constant KD = 39 nM and a capacity of n = 0.2 pmole/crista; the second had a KD = 16 microM and a capacity of n = 11 pmoles/crista. Aminoglycosides competed with uptake of gentamicin in the order netilmicin greater than or equal to neomycin greater than tobramycin, and polyamines competed in the order spermine greater than spermidine greater than putrescine; glucosamine and the basic amino acids lysine and asparagine were ineffective, excluding the participation of glucose and basic amino acid transport systems in gentamicin uptake. These results along with direct comparisons with some characteristics of putrescine uptake suggest that aminoglycoside and polyamine transport have common features in the crista ampullaris.