Escherichia coli is able to grow at increased NaCl concentrations that provides an increase in medium osmolarity and cellular Na+ content. The addition of 0.5 M NaCl to the growth medium led to a substantial decrease in growth rate during anaerobic fermentation on glucose at pH of 7.3 or 9.0. This inhibitory effect of 0.5 M NaCl was at least threefold stronger than that seen under aerobic conditions, and stronger than equivalent concentrations of sucrose, KCl, or potassium glutamate under anaerobic conditions. Further, proline was found to stimulate the growth rate at high NaCl concentration under anaerobic and to a lesser extent, under aerobic conditions. Wild-type cells and mutants having a functional NhaA or ChaA alone grown under anaerobic conditions at pH 9.0 and subsequently loaded with Naf were shown to extrude Na+ at a rate that were lower than the extrusion rate reported for appropriate aerobically grown bacteria (Sakuma et al. [1998] Biochim Biophys Acta 1363:231-237). The growth rate and Na+ extrusion activity of a mutant having a functional NhaA were similar to that of the wild type and higher than that of a mutant with an active ChaA. A mutant defective for both NhaA and ChaA was unable to grow under anaerobic conditions at pH 9.0 in the presence of 0.15 M Na+. It is suggested that the observed strong inhibition in the growth of E. coli during fermentation under anaerobic conditions in the presence of increased NaCl concentration could be due to a decrease in Naf extrusion activity.