Our knowledge on the absorption of folate is incomplete. The deconjugation process as a possible limiting factor in the absorption of folates was investigated. The study also attempted to validate the use of the area under the serum response curve (AUC) from food compared with folic acid as a proxy variable for food folate bioavailability. Folate absorption was determined in healthy ileostomy volunteers (n 11) using a single-dose short-term protocol. In a randomised crossover design, volunteers received spinach meals and a supplement. Based on analysis of test meals and ileostomy effluents, there was no difference in folate absorption between spinach with a mono-:polyglutamate ratio 40:60 and the same spinach with a 100:0 ratio. The absolute absorption of spinach folate (79 %) calculated from the difference between folate intake and folate content of ileostomy effluents was approximately equal to the relative absorption (81 %) calculated from the AUC after consumption of spinach meals in relation to the AUC after consumption of the folic acid supplement. We conclude that the deconjugation process is not a limiting factor in the absorption of spinach folates. Comparison of AUC of food folate v. folic acid in a short-term protocol may be suitable for assessing food folate bioavailability.