Ethambutol (EMB) concentrations that kill Mycobacterium tuberculosis in vitro accumulated in squirrel monkey tissues and cells known to be sites of tubercular infections. After oral administration of a clinically relevant 25 mg/kg dose, the whole-body distribution and intracellular localization of EMB were studied by radioautography. Tissue concentrations of drug were assayed by radiochemical and microbiological methods. The EMB was distributed rapidly and widely to most body tissues including lung and localized within pulmonary alveolar and axillary lymph node macrophages. The EMB in lung at 2 and 5 h after drug administration was markedly higher than the corresponding plasma concentrations and exceeded concentrations that are bactericidal in vitro for tubercle bacilli. These observations may help explain the early bactericidal activity of EMB in humans. Similarities in plasma and tissue concentrations of the drug in both species suggest the usefulness of the squirrel monkey as a model for the use of EMB in both species suggest the usefulness of the squirrel monkey as a model for the use of EMB in humans.