In vivo isotope-fractionation factors were determined for hydrogen and oxygen between plasma water samples and samples of urine, saliva, respiratory water vapor, and carbon dioxide in 20 normal adults. The isotope-fractionation factors ranged from 0.944 to 1.039 for 2H in breath water vapor and for 18O in breath CO2, respectively. When corrected for isotope fractionation, the 2H- and 18O-dilution spaces determined from urine, saliva, respiratory water, and CO2 were within -0.10 +/- 1.09 kg (mean +/- SD, n = 60) and 0.04 +/- 0.68 kg (n = 80), respectively, of the values determined from plasma. In the absence of these corrections, we observed a 6% overestimation of 2H-dilution space and a 1% overestimation of 18O-dilution space from the use of respiratory water values. A 4% underestimation of the 18O-dilution space was observed for breath CO2 without correction for isotope fractionation.