Conditions which induce a transmembrane electrical potential, positive with respect to the inside of membrane vesicles, result in a substantial (4-12-fold) stimulation of the activity of membrane-associated β-glucan synthetases in a membrane preparation derived from the developing cotton (Gossypium hirsutum L.) fiber. Induction of electrical potentials which are negative with respect to the inside of the membrane vesicle results in little or no stimulation of β-glucan synthesis. Those products whose synthesis is stimulated are mainly β-1,3-glucan, but there is also a considerable increase in β-1,4-glucan. No α-1,4-glucan (starch) was detected in the reaction products. A transmembrane pH gradient was found to have no effect on β-glucan synthesis. The results indicate that a transmembrane electrical potential can influence, either directly or indirectly, the activity of membrane-associated polysaccharide synthetases.