Elevated plasma lipid and nonesterified fatty acid concentrations reduce insulin-mediated glucose disposal in skeletal muscle. Cultured myoblasts from 21 subjects were studied for rates of palmitate oxidation and the effect of palmitate on glycogen synthase activity at the end of an 18-h incubation in serum- and glucose-free media. Oxidation rates of 40 µM palmitate in cultured myoblasts correlated with the fasting glucose (r = 0.71, P = 0.001), log fasting insulin (r = 0.52, P = 0.03), and insulin-mediated glucose storage rate (r = 0.50, P = 0.04) of the muscle donors. Myoblast glycogen synthase activity can be regulated by 240 µM palmitate, but the changes are associated with the basal respiratory quotient and not with the insulin resistance of the muscle donor. These results indicate that myoblasts producing elevated palmitate oxidation rates in vitro can be used to identify skeletal muscle abnormalities which are primary contributors to insulin resistance in vivo. Effects of 240 µM palmitate on myoblast glycogen synthase activity appear to be mechanistically different from the relationship between myoblast palmitate oxidation rates and insulin resistance of the muscle donor.