Mutations in the MTR gene, which encodes methionine synthase on human chromosome 1p43, result in the methylcobalamin deficiency G (cblG) disorder, which is characterized by homocystinuria, hyperhomocysteinemia, and hypomethioninemia. To investigate the molecular basis of the disorder, we have characterized the structure of the MTR gene, thereby identifying exon-intron boundaries. This enabled amplification of each of the 33 exons of the gene, from genomic DNA from a panel of 21 patients with cblG. Thirteen novel mutations were identified. These included five deletions (c.12-13delGC, c.381delA, c.2101delT, c.2669-2670delTG, and c.2796-2800delAAGTC) and two nonsense mutations (R585X and E1204X) that would result in synthesis of truncated proteins that lack portions critical for enzyme function. One mutation was identified that resulted in conversion of A to C of the invariant A of the 3' splice site of intron 9. Five missense mutations (A410P, S437Y, S450H, H595P, and I804T) were identified. The latter mutations, as well as the splice-site mutation, were not detected in a panel of 50 anonymous DNA samples, suggesting that these sequence changes are not polymorphisms present in the general population. In addition, a previously described missense mutation, P1173L, was detected in 16 patients in an expanded panel of 24 patients with cblG. Analysis of haplotypes constructed using sequence polymorphisms identified within the MTR gene demonstrated that this mutation, a C-->T transition in a CpG island, has occurred on at least two separate genetic backgrounds.