Objectives To determine reference values for serum nitric oxide metabolites (nitrite + nitrate = NO x ) concentrations in adult subjects. Design and methods Serum NO x concentration was measured, using the Griess method, in 694 non-smoking apparently healthy subjects, randomly selected from a population-based study. The International Federation of Clinical Chemistry guidelines and the robust method were used for determining reference values. Results The 95% reference values for serum NO x concentration and serum NO x /creatinine ratio were 11.5 to 76.4 μmol/L and 0.111 to 0.729 in men and 10.1 to 65.6 μmol/L and 0.121 to 0.777 in women, respectively. With increasing body mass index, upper limits of serum NO x and the NO x /creatinine ratio increased in women and decreased in men. Serum NO x levels above upper limits predicted both diabetes and metabolic syndrome in women. Conclusions This study reports the first set of reference values for serum NO x concentration and NO x /creatinine ratio in a relatively large healthy non-smoking population.