It is important to be able to evaluate vitamin status correctly at any age, but this is especially vital during adolescence since there are higher requirements for healthy growth and development. However, there are no currently available B-vitamin reference values for healthy adolescents. The aim of the present study is to assess the vitamin B status in European adolescents in order to contribute to the development of reference values for selected B-vitamins and total homocysteine (tHcy). Within the HELENA (Healthy Lifestyle in Europe by Nutrition in Adolescence) cross-sectional study, a sub sample of 1051 (499 males, 552 females) adolescents from ten European cities aged 12.5 to 17.49 were analyzed for fasting plasma folate (PF), red blood cell (RBC) folate, serum cobalamin (Cbl), holo-transcobalamin (Holo-TC), Vitamin B6 (PLP), and tHcy. The level of significance was set at P < .05. Following the current cut-off for adults, 2% had low Cbl and 5% had low holo-TC concentrations. Low concentrations of both PF and RBC folate were identified in 10%. Five percent had PLP concentrations <20 nmol/L and 20% <30 nmol/L. Moreover, 5% had high tHcy; median values for the whole sample were: PF 16.0 nmol/L, RBC folate 721.9 nmol/L, Cbl 319 pmol/L, Holo-TC 57.8 pmol/L, and tHcy 6.7 μmol/L. Females had significantly higher median Cbl but lower PLP and tHcy concentrations (P < .01). THcy increased (P < .001) and PF (P < .001) concentrations decreased across age categories. Subjects showed significantly higher tHcy values at the fifth percentile of PF, corresponding with 7.5 nmol/L. Sex and age had an influence on most of the studied biomarkers and should be taken into account. The HELENA percentile distribution is consistent with data from smaller studies and could be used as reference value to characterize B-vitamin status of European adolescents.