Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by impaired insulin-stimulated glucose uptake into glycogen. Both biochemical and genetic data have implicated glycogen synthase as a candidate for the genetic predisposition to diabetes. To test this hypothesis, we isolated cosmid clones containing genomic DNA for the glycogen synthase (GSY) gene and identified a region of 20 GT repeat units in a clone that extended 15 kilobases 3' to the gene. This region was highly polymorphic with nine alleles (heterozygosity 0.74). With the use of this polymorphism, the GSY was mapped on chromosome 19q between markers D19S217 and D19S210 and at theta = 0.036 from the histidine-rich calcium-binding protein (HRC) locus. Linkage to GSY was rejected under multiple models with logarithm of odds (LOD) scores of -1.36 to -5.22. In contrast, we could not reject linkage under dominant and intermediate (additive) models for the HRC locus (maximum LOD scores 1.51 and 1.54), despite the close proximity to GSY. Multipoint analysis of NIDDM versus GSY and HRC placed the putative diabetes locus centromeric to HRC and away from GSY. Furthermore, analysis of the previously associated Xba I polymorphism suggested neither linkage nor sib-pair sharing. We conclude that mutations of the GSY gene are unlikely to play a major role in the predisposition to NIDDM in our families. However, we cannot exclude a modifying role in a polygenic disorder or an important role in some families. The moderately positive LOD scores near the HRC locus suggest a need for evaluation of this region in additional NIDDM families.