A primary human skeletal muscle culture (HSMC) system, which retains cellular integrity and insulin responsiveness for glucose transport was employed to evaluate glucose transport regulation. As previously reported, cells cultured from non-insulin-dependent diabetic (NIDDM) subjects displayed significant reductions in both basal and acute insulin-stimulated transport compared to nondiabetic controls (NC). Fusion/differentiation of NC and NIDDM HSMC in elevated media insulin (from 22 pM to 30 microM) resulted in increased basal transport activities but reduced insulin-stimulated transport, so that cells were no longer insulin responsive. After fusion under hyperinsulinemic conditions, GLUT1 protein expression was elevated in both groups while GLUT4 protein level was unaltered. Fusion of HSMC under hyperglycemic conditions (10 and 20 mM) decreased glucose transport in NC cells only when combined with hyperinsulinemia. Hyperglycemia alone down-regulated transport in HSMC of NIDDM, while the combination of hyperglycemia and hyperinsulinemia had greater effects. In summary: (a) insulin resistance of glucose transport can be induced in HSMC of both NC and NIDDM by hyperinsulinemia and is accompanied by unaltered GLUT4 but increased GLUT1 levels; and (b) HSMC from NIDDM subjects demonstrate an increased sensitivity to impairment of glucose transport by hyperglycemia. These results indicate that insulin resistance in skeletal muscle can be acquired in NC and NIDDM from hyperinsulinemia alone but that NIDDM is uniquely sensitive to the additional influence of hyperglycemia.