The role of calmoudulin in control of carbohydrate metabolism in the absence and presence of insulin in isolated mouse soleus muscle was investigated. The calmodulin antagonist CGS 9343B had no effect on basal glycogen synthase activity, the contents of high energy phosphates, glucose-6-P, or glycogen synthesis. However, CGS 9343B inhibited the basal rates of 2-deoxyglucose uptake and 3-O-methylglucose transport by 30% (p < 0.05) and 40% (p < 0.001), respectively. Insulin activated glycogen synthase by almost 40% (p < 0.01) and this increase was not altered in the presence of CGS 9343B. Insulin increased the muscle content of glucose-6-P (approximately equal to 2-fold), as well as glycogen synthesis (approximately equal to 8-fold), 2-deoxyglucose uptake (approximately equal to 3-fold), and 3-O-methylglucose transport (approximately equal to 2-fold), and these increases were inhibited by CGS 9343B. In additional experiments on isolated rat epitrochlearis muscle, it was found that the hypoxia-mediated activation of 3-O-methylglucose transport was also inhibited by CGS 9343B. These data demonstrate that: 1) hexose transport, both in the absence and presence of external stimuli (insulin and hypoxia), requires functional calmodulin; and 2) insulin-mediated activation of glycogen synthase does not require functional calmodulin, nor can it be accounted for by increases in glucose transport or glucose-6-P.