The pharmacokinetics of gentamicin, tobramycin, and amikacin in inner ear fluids, serum, cerebrospinal fluid, and the compartments of the eye were studied and compared in guinea pigs. The concentrations of antibiotic were determined by microbiologic methods and were confirmed by the use of 14C-labeled gentamicin. Retention was clearly demonstrated in perilymph, in which the half-lives of gentamicin, tobramycin, and amikacin were 12, 11, and 10 hr, respectively. The concentrations of drug in perilymph were symmetrical and were many times higher than the concentrations of antibiotic in the brain. There was no difference between the concentration of drug in endolymph and that in perilymph. A linear relation between concentrations in the perilymph and the dosage of gentamicin was ascertained. Long-term treatment did not influence the pharmacokinetics of the three antibiotics in the inner ear. However, increased levels of drug in the inner ear in animals with uremia and in some animals with otitis media explained the increased ototoxicity that occurs in treatment of these two conditions. Suboccipital puncture and diuresis did not change the concentrations of aminoglycoside antibiotics in the inner ear. Antibiotics applied locally in the middle ear had high degrees of ototoxicity.