Calsequestrin (CASQ)1 is involved in intracellular storage and release of calcium, a process that has been shown to mediate glucose transport in muscle. Its gene, CASQ1, is encoded on chromosome 1q21, a region that has been linked to type 2 diabetes in the Amish and several other populations. We screened all 11 exons, exon-intron junctions, and the proximal regulatory region of CASQ1 for mutations. We detected four novel single nucleotide polymorphisms (SNPs) (-1470C-->T, -1456delG, -1366insG, and 593C-->T). Ten informative SNPs within CASQ1 were genotyped in Amish subjects with type 2 diabetes (n = 145), impaired glucose tolerance (n = 148), and normal glucose tolerance (n = 358). Rs2275703 and rs617698 in introns 4 and 2 were significantly associated with type 2 diabetes (P = 0.008 and 0.04, respectively); three other SNPs showed borderline evidence for association to type 2 diabetes (P = 0.076-0.093). Furthermore, in nondiabetic subjects (n = 754), both rs2275703 and rs617698 were significantly associated with glucose area under the curve during an oral glucose tolerance test (P = 0.035 and 0.013, respectively). Haplotype analysis suggested that no haplotype could explain these associations better than rs2275703. These findings, coupled with similar findings in Utah Caucasians, suggest that sequence variation in CASQ1 may influence risk of type 2 diabetes.