It has been suggested that vitamin D₂ is not very prevalent in the human food chain. However, data from a number of recent intervention studies suggest that the majority of subjects had measurable serum 25-hydroxyvitamin D₂ (25(OH)D₂) concentrations. Serum 25(OH)D₂, unlike 25(OH)D₃, is not directly influenced by exposure of skin to sun and thus has dietary origins; however, quantifying dietary vitamin D₂ is difficult due to the limitations of food composition data. Therefore, the present study aimed to characterise serum 25(OH)D₂ concentrations in the participants of the National Adult Nutrition Survey (NANS) in Ireland, and to use these serum concentrations to estimate the intake of vitamin D₂ using a mathematical modelling approach. Serum 25(OH)D₂ concentration was measured by a liquid chromatography-tandem MS method, and information on diet as well as subject characteristics was obtained from the NANS. Of these participants, 78.7 % (n 884) had serum 25(OH)D₂ concentrations above the limit of quantification, and the mean, maximum, 10th, 50th (median) and 90th percentile values of serum 25(OH)D₂ concentrations were 3.69, 27.6, 1.71, 2.96 and 6.36 nmol/l, respectively. To approximate the intake of vitamin D₂ from these serum 25(OH)D₂ concentrations, we used recently published data on the relationship between vitamin D intake and the responses of serum 25(OH)D concentrations. The projected 5th to 95th percentile intakes of vitamin D₂ for adults were in the range of 0.9-1.2 and 5-6 μg/d, respectively, and the median intake ranged from 1.7 to 2.3 μg/d. In conclusion, the present data demonstrate that 25(OH)D₂ concentrations are present in the sera of adults from this nationally representative sample. Vitamin D₂ may have an impact on nutritional adequacy at a population level and thus warrants further investigation.