The hyc operon of Escherichia coli encodes the H2-evolving hydrogenase 3 (Hyd-3) complex that, in conjunction with formate dehydrogenase H (Fdh-H), constitutes a membrane-associated formate hydrogenlyase (FHL) catalyzing the disproportionation of formate to CO2 and H2 during fermentative growth at low pH. Recently, an operon (hyf) encoding a potential second H2-evolving hydrogenase (Hyd-4) was identified in E. coli. In this study the roles of the hyc- and hyf-encoded systems in formate-dependent H2 production and Fdh-H activity have been investigated. In cells grown on glucose under fermentative conditions at slightly acidic pH the production of H2 was mostly Hyd-3- and Fdh-H-dependent, and Fdh-H activity was also mainly Hyd-3-dependent. However, at slightly alkaline pH, H2 production was found to be largely Hyd-4, Fdh-H and F0F1-ATPase-dependent, and Fdh-H activity was partially dependent on Hyd-4 and F0F1-ATPase. These results suggest that, at slightly alkaline pH, H2 production and Fdh-H activity are dependent on both the F0F1-ATPase and a novel FHL, designated FHL-2, which is composed of Hyd-4 and Fdh-H, and is driven by a proton gradient established by the F0F1-ATPase.