One of the specific inhibitors of calmodulin action, trifluoperazine, blocked the stimulating action of insulin on 2-deoxyglucose uptake and glucose metabolism. The inhibitory effect of insulin on lipolysis was not altered by the drug. The active (insulin-stimulated) state and the basal state of lipogenesis were inhibited half-maximally at 80 and 550 microM trifluoperazine, respectively. 2-Deoxyglucose uptake was inhibited half-maximally at a trifluoperazine concentration of 70 microM. Other less potent calmodulin inhibitors also inhibited glucose metabolism in fat cells but in a nonspecific manner. The inhibition was noncompetitive and was not altered in Ca2+- free medium. The stimulating activity of wheat germ agglutinin and of sodium vanadate were also inhibited by trifluoperazine. The dose-dependent inhibitions were indistinguishable whether the active (stimulated) state was produced by insulin, wheat germ agglutinin, or vanadate. The data indicate that a late event in the sequence that ultimately leads to enhanced glucose transport activity in fat cells is specifically inhibited by trifluoperazine. The possible involvement of calmodulin or another related Ca2+-dependent regulatory protein in the exocytic (fusion) reaction that recruits glucose-transport activity from storage sites to the plasma membranes is discussed.