The intestinal absorption of folate occurs at the monoglutamyl level, and an important measure of food folate bioavailability is how much folate from the food reaches the intestinal sites in forms that can readily be absorbed. In the absence of protecting agents, e.g., vitamin C and reduced thiols, many labile folates may be lost during cooking and during residence in the acid-peptic milieu of the stomach. On the other hand, the presence of polyglutamyl folate necessitates the action of intestinal hydrolases, which could be affected by food constituents. In this study, we developed an in vitro assay for the determination of an index of food folate availability. The index of folate availability in this study was defined as that proportion of folate that has been identified as monoglutamyl derivatives after tests for stability and susceptibility to an enzymatic hydrolysis. The index of folate availability varied widely among foods. The highest index was for egg yolk (72.2%), followed by cow's livers (55.7%), orange juice (21. 3%), cabbage (6.0%), lima beans (4.5%) and lettuce (2.9%). Yeast folate had the lowest index (0.3%). The availability indices generated by this study correlate with the indices of the bioavailability of the corresponding food folate observed in earlier studies, R2 = 0.529 (P = 0.068). Additional information is required on the bioavailability of other food products to test the usefulness of this in vitro approach for assessing food folate availability.