Effects of long-term inhalation of 100 per cent oxygen on the microbial flora of the rat larynx, trachea, and large intestine were studied. Rats were kept 14 days in an atmosphere of 100 per cent oxygen after being conditioned to high oxygen concentrations by exposure to three cycles of 100 per cent oxygen (two days) alternating with 40 per cent oxygen (two days). Controls were kept under similar conditions in normal atmosphere. Rats were sacrificed, and at necropsy laryngotracheal swabs and fecal material from the large intestine were obtained and cultured for bacteria and fungi.Streptobacillus moniliformis, the predominant microorganism in the upper tracheas of controls, was not isolated from the oxygen-treated rats. Alpha-hemolytic streptococcus andStaphylococcus albuswere present in control rats, but were found less frequently in rats exposed to oxygen.PseudomonasandProteus, infrequently isolated from controls, were predominant and sometimes the only microorganisms isolated from oxygen-treated rats. The data indicate that prolonged exposure of the rat to 100 per cent oxygen shifts the microbial flora in the upper respiratory tract from mainly gram-positive to mainly gram-negative bacteria. In contrast, there was no significant difference between the microbial flora in large intestines of control and oxygen-treated rats. The possibility that similar changes may occur in man should be considered when prolonged oxygen therapy is contemplated.