The influence of high luminal PO2 on Colonic anaerobes was investigated by chronically perfusing the colon of unrestrained rats with O2 or N2. A luminal PO2, of approximately 700 mm Hg did not significantly influence the fecal concentration of total anaerobes recovered on rabbit blood agar or of Bifidobacterium sp. and Bacteroides sp. recovered on selective media. However, the O2 infusion reduced the rat's excretion of CH4 (a metabolic product of anaerobic bacteria) to about 5% of the control value, although the feces of O2-treated rats produced CH4 normally. This study suggests that the production of metabolites by the Colonic bacteria may sometimes be more accurately studied in situ rather than in culture. Measurement of volatile bacterial metabolites in breath may provide a simple means of studying the in situ metabolism of a variety of colonic bacteria in man.