Fumarase deficiency is a rare autosomal recessive disorder of the citric acid cycle causing severe neurological impairment. The cDNA for both the rat and human enzymes has been cloned previously and shown to encode a coding region of 1.46 kb. To scan for mutations in fumarase-deficient patients we amplified the coding region of fumarase from fibroblast/lymphoblast cDNA employing the oligonucleotide primers designed from the published human and rat cDNA sequence. We then directly sequenced the polymerase chain reaction product. In seven unrelated patients, we detected four missense mutations (A265T, D383V, F269C, K187R), a nonsense mutation (W458X), a 3-bp AAA insertion that introduces an additional lysine residue at codon 435, and a spontaneous new mutation resulting in a 74-bp deletion (66del74). Seven at-risk pregnancies were monitored with one prenatal diagnosis of fumarase deficiency by molecular analysis and favorable outcome of the other pregnancies as predicted by enzyme assay of cultured fetal cells or molecular analysis.