Lysis of exponential cultures of B. subtilis follows the addition of reagents that dissipate either the electrical or pH gradients of cellular membranes. Stationary-phase cells or cultures that have been inhibited in division by macromolecular-synthesis inhibitors also lyse when uncoupling agents or ionophores are added to the growth medium. Autolysis occurs after brief starvation for a carbon source. Protoplasts are unaffected by azide or other lysis-inducing agents. Electron-donating agents, such as phenazine methosulfate and ascorbate, are effective in retarding autolysis. The addition of an oxidizable carbon source to starved and lysing cultures prevents their autolysis. These results suggest that cellular lysis in B. subtilis and energized membrane are tightly coupled. The fluorescence intensity and the wavelength of maximal fluorescence of 8-anilino-1-naphthalene sulfonic acid, when added to bacterial suspensions, appear to be qualitatively related to the rate of cell lysis. Analyses show that ATP limitations are probably not involved in the elicitation of lysis by ionophores, uncoupling agents or starvation. Measurements of protonmotive forces in the lysis-prone cells suggest that a threshold force of more than 85 mV may be required to maintain cellular integrity. Lipoteichoic acids, polyelectrolytes such as dextran sulfate or phospholipids do not modify the rate of cellular lysis when added to suspensions containing azide or other reagents that eliminate transmembrane protonmotive forces. We interpret the results to suggest that the in vivo control of autolysin activity in B. subtilis is related to the energized membrane