The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na+]in) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H+) or sodium ions (Na+), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H+- and Na+-driven components that functions as a sodium motor. Changing external sodium concentration ([Na+]ex) in the range 1-85 mM resulted in changes in [Na+]in between 5-14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na+]in and [Na+]ex, and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2-3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.