Background: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size-and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP. Methods: Flow cytometry and spectrofluorometry were used to detect red (488 = G600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC 2 (3)), which, in the monomeric state, is normally green (488 = 530 nm) fluorescent. Results: In bacteria incubated with 30 ยตM dye, aggregate formation increases with the magnitude of the interiornegative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from ุ50 mV through ุ120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4ยฐC and washing in glucose-free medium. Conclusions: The ratiometric technique for MP estimation using DiOC 2 (3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.