Objective: The aim of this study was to investigate whether milk fortified with folic acid enhances the folate status of humans and whether the presence of folate-binding proteins (FBP) in pasteurised milk affects the bioavailability of folic acid from fortified milk. In untreated and pasteurised milk, folate occurs bound to FBP, while FBP is (partly) denatured in ultra-high-temperature (UHT)-treated milk. The effect of FBP on folate bioavailability is still unclear. Design, subjects and setting: Healthy, free-living subjects (n=69) aged 18-49y participated in a 4-week double-blind, placebo-controlled dietary intervention study. Intervention: In addition to a fully controlled diet, the subjects consumed each day 500 ml of pasteurised or UHT milk, either fortified or not with 200 μg folic acid. Results: Consumption of fortified milk increased folate concentrations in serum and in red blood cells (RBQ by 6.6-7.0 nmol/l (P<0.001) and 32-36nmol/l (P<0.01), respectively. Similarly, plasma homocysteine concentrations were lowered 0.88-0.89 μmol/l (P = 0.001) in subjects who consumed fortified milk. The bioavailability of folic acid from pasteurised milk relative to that of folic acid from UHT milk was 74-94% (NS), depending on the parameter used. Conclusions: Milk fortified to supply an additional 200 μg of folic acid/s substantially increased folate status, and decreased plasma total homocysteine concentrations in young, healthy subjects. Milk is therefore a suitable matrix for fortification to enhance the folate status in humans. No significant effect of endogenous FBP was found on the bioavailability of folic acid from milk. © 2005 Nature Publishing Group. All rights reserved.