The unambiguous synthesis of 2 folate analogues, in which the 10-amino group of folic acid was replaced with O2, was described. The synthetic sequence employed commercially avilable methyl p-hydroxybenzoate and N-(2,3-epoxypropyl)phthalimide as starting materials. Both 10-oxafolic acid (1) and 10-oxaaminopterin (2) showed potent antifolate activity when tested against folate-requiring organisms [Streptococcus faecium]. Compound 2 was a very powerful inhibitor of 3'',5''-dichloromethopterin resistant Lactobacillus casei dihydrofolate reductase [EC 1.5.1.3]. The activity was comparable to that of methotrexate while the 4-hydroxy analogue did not show inhibition. 7,8-Dihydro-10-oxafolic acid failed to show any substrate activity to this enzyme and did not inhibit the enzymatic reaction when used with an equimolar concentration of the natural substrate. Folate analogues are often used as chemotherapeutic agents in neoplastic diseases.