Even if in the absence of clinical folate deficiency, providing supplemental folic acid in the periconceptional period lowers the risk that the infant will have a neural tube defect (NTD). Findings that antifolate receptor antiserum leads to resorption of embryos as well as malformations in rats suggested that autoantibodies against these receptors could dispose to defects. The study group included 12 women who were, or had been, pregnant with an affected fetus and 24 control women (20 with a normal current or prior pregnancy and 4 nulligravidas). Serum specimens were analyzed for antifolate receptor autoantibodies by incubating samples with purified human placental folate receptors radiolabeled with 3H-folic acid. Serum and autoantibodies isolated from serum were incubated with placental membranes, ED27 human placental cells, and KB cells, a human epidermoid carcinoma cell line, which express folate receptors. No significant demographic or clinical differences were found between the index subjects and control subjects. Serum antibodies against folate receptors were identified in 9 of the 12 index women (75%). Two of them were currently pregnant with a fetus exhibiting a NTD, whereas 7 had a history of such a pregnancy. Two of 20 control women (10%) had a current or previous infant with a NTD. The mean value for folate receptors bound by antiantibody was significantly greater in the index subjects. Only sera testing positive for autoantibody-blocked folic acid binding to placental membrane receptors, ED27 cells, and KB cells incubated at 4°C. Autoantibodies also blocked the uptake of 3H-folic acid by KB cells incubated at 37°C. No anticardiolipin, antisingle-stranded DNA, or antithyroid autoantibodies were identified in sera from index subjects. Autoantibodies against folate receptors are present in sera from women whose pregnancies are complicated by a NTD. It remains to be determined whether these autoantibodies are pathogenic.