Objective: To analyse the Nutritional Knowledge Test (NKT) using Item Response Theory (ITR) analysis and to assess the construct validity of the Nutritional Knowledge Scale (NKTS) and its associations with adolescent food group consumption and nutritional biomarkers. Design: Cross-sectional study. Setting: Multicentre investigation conducted in ten European cities. Participants: Adolescents aged 12.5-17.5 years (n 3215) who completed over 75 % of the NKT. Results: Factor analysis indicated that the NKT can be analysed with a one-dimensional model. Eleven out of twenty-three items from the NKT presented adequate parameters and were selected to be included in the NKTS. Nutrition knowledge was positively associated with consumption of fruits, cereals, dairy products, pulses, meat and eggs, and fish, as well as with blood concentrations of vitamin C, beta-carotene, n-3 fatty acids, holo-transcobalamin, cobalamin and folate; nutrition knowledge was negatively associated with intake of olives and avocado, alcohol and savoury snacks. Conclusions: The NKTS assessed nutritional knowledge adequately and it is proposed as a new tool to investigate this subject in future studies.