Stable isotope dilution assays were developed for the quantification of the folate vitamers 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, tetrahydrofolate, 10-formylfolate and pteroylglutamic acid in food samples by using deuterated isotopomers as internal standards. Vitamers and their labeled analogues were analyzed simultaneously by HPLC/MS/MS using selected reaction monitoring, which allowed a higher specificity than other methods published previously. Sample preparation involved treatment by protease in sequence with alpha-amylase and rat serum deconjugase, followed by anion exchange chromatography. The detection limits for 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, tetrahydrofolate, 10-formylfolate and pteroylglutamic acid were found to be 0.5, 1.2, 1.5, 0.6 and 2.6 microg/100 g fresh weight, respectively. Using the new method, folate contents were determined in meat, cereals, and vegetables. Data were in good agreement with literature data, except results for broccoli, which were much lower than reported in previous studies.