Summary. The availability of five different folate derivatives: tetrahydropteroylglutamate (H4PteGlu), 5-formyltetrahydropteroylglutamate (5-CHO-H4PteGlu), 5-methyltetrahydropteroylglutamate (5-CH3 -H4PteGlu), pteroyltriglutamate (PteGlu3) and pteroylheptaglutamate (PteGlu7) and folates in 12 natural food items: orange juice, romaine lettuce, egg yolk, banana, cooked lima beans (dry and frozen), liver, brewer's yeast, cabbage (raw and cooked), defatted soybean, and wheat germ were measured in human subjects. The method used for this study was based on the measurement of urinary excretion of folate by microbiological assay using Lactobacillus casei (L. casei). Each subject was maintained in a ‘folate-saturated’ condition by oral administration of 2.0 mg of pure pteroylmonoglutamate (PteGlu) on alternate days and a response curve relating folate intake with urinary folate excretion was established. Urinary folate excretion following administration of synthetic folate derivatives and food samples containing microbiologically assayed quantities of folates were measured and the folates which had been absorbed were estimated by reference to the response curve previously established with PteGlu. We have considered this to be an index of the ‘availability’ of synthetic folates and/or naturally occurring folates in foods. The results indicated that pure synthetic folate derivatives are almost as available as PteGlu. Among the 12 food items, banana, lima beans, liver and bewer's yeast contained relatively highly available folates. Folate availability was poor in orange juice, romaine lettuce, egg yolk, cabbage, defatted soybean and wheat germ.