Carbon dioxide production, water production, and energy expenditure (EE) determined with 2H2(18)O were validated using a room calorimeter. Nine human subjects (5 males, 4 females) resided in the calorimeter for five to seven consecutive 23.5-h days. Daily carbon dioxide and water production and EE were averaged from calorimeter data and compared with 2H2(18)O results for the same period and for the following week. Isotope concentrations were measured by isotope ratio mass spectroscopy (18O) and infrared spectrophotometry (2H). Isotope clearance rates and pool sizes were determined by two multipoint methods and the two-point method. No significant differences were measured between calorimetry and 2H2(18)O results for the period within the chamber [multipoint A.M. urine 1.55% +/- 2.57 (SD) %, multipoint A.M. + P.M. urine 0.98% +/- 8.19%, two-point 1.59% +/- 4.50%]. Free-living EE was 13% +/- 7% greater. The results indicate that the 2H2(18)O method as applied is a valid technique for measuring EE, that multipoint calculation methods can be as precise as the two-point method, and that EE was greater in free-living than in calorimeter-confined conditions.