A novel method based on the intracellular conjugation of the fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester (cFSE) was developed to determine the intracellular pH of bacteria. cFSE can be taken up by bacteria in the form of its diacetate ester, 5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester, which is subsequently hydrolyzed by esterases to cFSE in the cytoplasm. When Lactococcus lactis cells were permeabilized with ethanol, a significant proportion of cFSE was retained in the cells, which indicated that cFSE was bound intracellularly. Unbound probe could be conveniently extruded by a short incubation of the cells in the presence of a fermentable sugar, most likely by exploiting an active transport system. Such a transport system for cFSE was identified in L. lactis, Listeria innocua, and Bacillus subtilis. The intracellular pH in bacteria can be determined from the ratio of the fluorescence signal at the pH-sensitive wavelength (490 nm) and the fluorescence signal at the pH-insensitive wavelength (440 nm). This cFSE ratio method significantly reduced problems due to the efflux of fluorescent probe from the cells during the measurement. Moreover, the method described was successfully used to determine the intracellular pH in bacteria under stress conditions, such as elevated temperatures and the presence of detergents.