The relationship of membrane potential to motility and chemotaxis of Bacillus subtilis has been tested by using the fluorescence of a cyanine dye as a probe of the potential. The dye fluorescence was found to be an indicator of membrane potential by correlation with triphenylmethylphosphonium ion distribution and with changes due to anaerobicity and ionophore addition. When the potential was sufficient for motility and constant over time, it was found that the absolute level of the potential did not affect the swimming behavior of the bacteria. Transient alteration of the membrane potential did, however, lead to changes in swimming behavior. Attractants were found to alter the swimming behavior of the bacteria without altering the membrane potential. Thus, change of the overall membrane potential of a normal B. subtilis is not required for chemotaxis, but such a change is sensed by the bacteria just as changing levels of attractants and repellents are sensed.