Despite its low natural folate concentration, milk is responsible for 10-15% of the daily folate intake in countries with a high dairy consumption. Milk products can be considered as a potential matrix for folate fortification, e.g., with synthetic folic acid, to enhance the daily intake of folate. In untreated milk, the natural folate, 5-methyltetrahydrofolate (5-CH 3-H4folate), is bound to folate-binding proteins (FBP). In this study, the extent of binding to FBP for folic acid and 5-CH 3-H4 folate was investigated in a dynamic in vitro model simulating human gastric passage. Protein binding of folic acid and 5-CH 3-H4 folate was characterized using gel-exclusion chromatography. Before gastric passage, folic acid and 5-CH3-H 4 folate were bound mainly to FBP (76-79%), whereas 7% was free. Folic acid remained bound to FBP to a similar extent after gastric passage. For 5-CH3-H4 folate, the FBP-bound fraction gradually decreased from 79 to 5% and the free fraction increased from 7 to 93%. Although folic acid enters the proximal part of the intestine bound to FBP, 5-CH 3-H4 folate appears to be present mainly as free folate in the duodenal lumen. The stability of FBP was similar in both folate/FBP mixtures, i.e., 70% of the initial FBP content was retained after gastric passage. This study indicated that FBP are partly stable during gastric passage but have different binding characteristics for folic acid and 5-CH 3-H4 folate in the duodenal lumen. This could result in different bioavailability from folic acid- and 5-CH3-H4 folate-fortified milk products.