INTRODUCTION: Total body water (TBW) assessment is a cornerstone in order to validate equations to estimate body composition components as fat mass and fat free mass. Deuterium dilution technique (D2DT) is considered the gold standard method to assess TBW, however in Spain there are not any reference data using D2DT. The knowledge of specific references for Spanish population must be a goal for the biological studies of Spanish people. OBJECTIVE: It was our aim to assess TBW using the D2DT in a sample of Spanish adolescents. METHODS: 224 specimens of urine from 150 adolescents (113 girls and 111 boys) were taken to analyze. A dose of 0.1 grams of 2D2O per Kg of body weight was administrated; afterwards it was followed by 4-hours stabilization period. The isotope (deuterium oxide (D2O)) dilution method was used to measure TBW with an isotope-ratio mass-spectrometer. Averages by sex and Tanner stages were calculated for %TBW and TBW. General lineal model were used to analyze differences and interactions between groups. RESULTS: %TBW was significantly different between boys and girls (58.98_5.59% vs 55.5_4.28%, P<0.001). Significant differences were found among 12 years and other groups of age (15, 16, 17 and 18; P<0.05) but not for maturation groups (P>0.05). CONCLUSIONS: This study has been the first in Spain, which have measured TBW in vivo using D2DT. These data can be useful to validate new methods and equations from bioimpedance analysis to estimate TBW.