The marked lability in cell-free preparations of the enzyme system involved in cellulose biosynthesis in most organisms studied led us to investigate factors responsible for loss of activity on cellular disruption. Previous studies have led to the suggestion that the existence of a transmembrane electrical potential (DeltaPsi) may be one factor responsible for maintaining an active system in intact cells. In this report, we show that dissipation of the DeltaPsi in metabolizing cells of Acetobacter xylinum results in severe inhibition of cellulose synthesis. The effect can be reversed by restoration of the DeltaPsi. Inhibition of cellulose biosynthesis by dissipation of the DeltaPsi can be observed under conditions in which no substantial impairment of energy metabolism occurs-i.e., under conditions in which a transmembrane pH gradient is of sufficient magnitude to maintain an adequate overall protonmotive force across the membrane. The inhibition of cellulose biosynthesis is specifically related to changes in the DeltaPsi, since the process can proceed normally in the absence of the pH gradient. These results support the suggestion that loss of the DeltaPsi on cellular disruption may be one of the factors responsible for the low capacity for cellulose synthesis in isolated membrane preparations and also raise the possibility that modulation of the DeltaPsi could be one means of regulating the rate of cellulose synthesis in vivo.