The half-life of gentamicin, tobramycin, kanamycin, and streptomycin, after a single subcutaneous injection, was measured in homogenates of lung, liver, and kidney in rats, and the results were compared with the half-life of these drugs in serum. Urinary excretion of antibiotics was also monitored. The half-life of the four drugs in serum and lung was approximately 30–35 min. Concentrations in the liver were regularly lower than concentrations in serum or lung. In renal tissue, gentamicin, tobramycin, and kanamycin had long half-lives of 109, 74, and 60 hr, respectively. Streptomycin, on the other hand, had a half-life of 4.6 hr. Separation of the kidneys into cortex and medulla showed that streptomycin was uniformly distributed, whereas 85% of the gentamicin, tobramycin, and kanamycin resided in the cortex. These antibiotics accumulate in the renal cortex, probably intracellularly, and have long half-lives.