Background: Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H2 production involves consumption of 2H+ , hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2–2.5) that are three pH units lower than the pH limit of growth (pH 5–6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms. Methods and Principal Findings: We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H2 to 2H+ . Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H2 production and consumption was tested using a H2-specific Clark-type electrode. Hyd-3-dependent H2 production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H2 consumption was maximal at alkaline pH. H2 production, was unaffected by a shift in external or internal pH. H2 production was associated with hycE expression levels as a function of external pH. Conclusions: Anaerobic growing cultures of E. coli generate H2 via Hyd-3 at low external pH, and consume H2 via Hyd-2 at high external pH. Hyd-3 proton conversion to H2 is required for acid resistance in anaerobic cultures of E. coli.