The objective of the study was to develop and validate a 21-item nutrition knowledge test to measure people's knowledge of the fat content of food-products. After pretesting and provisional development, the test was administered twice to study test-retest reliability. Furthermore, various sub-populations with expected differences in nutrition knowledge completed the test in order to study the construct validity of the questionnaire. The subpopulations consisted of lay-people (N = 81), students of human nutrition and dietetics (N = 108), and professional experts (N = 79) on human nutrition. The internal consistency and uni-dimensionality of the test were determined by calculating the KR-20 statistic and the log-likelihood ratio statistic for the Rasch model. Pearson's correlation and gross misclassification between T1 and T2 were calculated to assess the test-retest reliability. Analysis of variance was used to test for differences in mean knowledge scores between subpopulations. Test-retest reliability was found to be sufficient (R = .85). The internal consistency was moderate (KR20 = .68). According to the Rasch model, two items had to be removed from the test before the log-likelihood ratio statistic of the Rasch model indicated that knowledge about the fat content of food products as assessed by the questionnaire is a uni-dimensional construct. The differences in mean knowledge scores between the subpopulations were significant (p < .01) and in the expected direction (experts > students > lay people). It can be concluded that the test is a reliable and valid instrument to measure knowledge about total fat content in food products and that the Rasch model is a comprehensive method to indicate the reliability of nutritional knowledge tests.