Skeletal muscle glycogen synthase (encoded by GYS1 on chromosome 19q13.3) is the rate-limiting enzyme in insulin-mediated non-oxidative glucose disposal. Our previous studies have demonstrated an impairment of insulin-stimulated GYS1 activities in insulin-resistant Pima Indians, and associations of non-insulin-dependent diabetes mellitus (NIDDM) with the GYS1 locus were reported recently in Finnish and Japanese populations. We have performed linkage and association analyses of GYS1 and seven additional DNA markers on 19q with NIDDM, and with parameters of insulin action in the Pima Indians. We have found a significant association of NIDDM with GYS1 genotypes (p = 0.009), and with common GYS1 alleles (p = 0.022) in the Pima Indians. We have performed a detailed comparative analysis of the GYS1 gene, mRNA, and protein product in insulin-sensitive and insulin-resistant Pima Indians. No mutations in GYS1 coding sequences were detected; nor did we find alterations of GYS1 mRNA expression or of its basal enzymatic activity in insulin-resistant Pima Indians. These results contrasted with a 25% reduction of immunoreactive protein in insulin-resistant subjects as detected by Western blotting with an antibody specific for the C-terminal end of GYS1 (t-test p = 0.024; Wilcoxon's rank-sum test, p = 0.04). Because no mutations were detected in the DNA encoding this epitope, the difference in immunoreactivity may reflect post-translational modification(s) of the protein rather than a difference in the gene itself, or it could have occurred by chance. We conclude that our data do not indicate alterations in the GYS1 gene as the cause for the observed association, and that a different locus near GYS1 may be the contributing genetic element.