Fabry disease (FD) is an X-linked inborn error of glycosphingolipid catabolism resulting from the deficient activity of the lysosomal glycosidase,α-galactosidase A (α-Gal A). In classically affected males with little, if any, α-Gal A activity, vascular disease of the heart, kidney, and brain leads to premature demise in adulthood. Previous studies identified different mutations in the α-Gal A gene causing FD and determined that most mutations were private. To further characterize the molecular lesions causing FD and for precise heterozygote detection, mutation analysis was performed by PCR-amplification and solid-phase direct sequencing. Eighteen new mutations were identified including 11 missense (Y86C, D92Y, C94Y, A97V, Y134S, G138R, A143T, S148R, D170V, C202Y, Y216D, one nonsense (W399X), and five small exonic insertions or deletions (613del9, 1057del2, 1074del2, 1212del3, and 1094ins1), which identified exon 7 as a region prone to gene rearrangements. In addition, a unique complex rearrangement was found in exon 2 resulting in L120X. This complex rearrangement involved a series of three small deletions in a 13 bp region (G GCA GAG CTC ATG) from nucleotide 216 to 228, which caused a frameshift and truncation at codon 72. The two mutations involving cysteines (C94Y and C202Y) occurred in residues that were conserved in 85% and 90%, respectively, of 20 homologues of α-Gal A and the related enzyme, α-Gal B, indicating their probable importance in the formation of intramolecular disulfide bridges in the enzyme polypeptide. Three of the missense mutations (Y86C, D92Y, C94Y) occurred in exon 2, in a highly conserved region presumed to contain residues in the active site. In addition, all three exon 2 missense mutations were highly conserved (80% to 95%). Other mutations that occurred at highly conserved (>75%) residues were Y134S, G138R, S148R, and D170V, indicating their importance in the activity and/or stability of the enzyme. The identification of these 18 mutations further documents the remarkable genetic heterogeneity of the α-Gal A lesions causing Fabry disease.