The relation of intracolonic gaseous tension to fecal microflora was investigated by mass spectrometric measurements of intracolonic O2 and CO2 in unanesthetized germfree, conventional, and gnotobiotic rats; ip measurements were obtained in rats whose colons became perforated accidentally; fecal bacterial flora and cecal size were also determined. Gnotobiotes were monoassociated with Escherichia coli, Bacteroides fragilis and Staphylococcus epidermidis, and were diassociated with E. coli plus B. fragilis and with Bacillus macerans plus an aerobic diphtheroid. Mean intracolonic Po2 in conventional rats (11.1 mmHg) was significantly lower than in germfree rats (12.8 mmHg); mean intracolonic Pco2 in conventionals (83.4 mmHg) was greater than in germfree rats (54.6 mmHg). Differences of Po2 values among all rats were slight. However, intracolonic Pco2 values were directly related to CO2 production by the normal intestinal flora, and were thus significantly lower in both germfree and gnotobiotic rats. Intraperitoneal tensions were independent of the status of rats, and the mean ip Po2 and Pco2 (38.9 and 49.1 mmHg, respectively) agreed with values in the literature. The enlarged cecum, characteristic of germfree rats, was also present in gnotobiotic rats. Counts of each bacterial species in feces of monoassociated rats were 10(8) to 10(10)/g; counts were progressively smaller in feces of diassociated rats and conventional rats (10(8) to 10(9)/g and 10(5) to 10(8)/g, respectively). Intracolonic gaseous tensions of CO2 clearly reflected the presence of a normal flora inconventional rats, and were inversely proportional to cecal size.