Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (Vm) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure Vm in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated Vm using the ratio of intracellular/extracellular dye concentration. We found Vm = -140 ± 14 mV in Escherichia coli at external pH 7.0 (pHex), decreasing to -85 ± 10 mV at pHex 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of Vm and intracellular sodium concentration. We were able to vary the SMF between -187 ± 15 mV and -53 ± 15 mV by varying pHex in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-μm- and 1-μm-diameter beads attached to Na +-driven chimeric flagellar motors varied linearly with V m. For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.