The nucleotide sequence has been determined for a twelve-gene operon of Escherichia coli designated the hyf operon (hyfABCDEFGHIR-focB). The hyf operon is located at 55.8-56.0 min and encodes a putative nine-subunit hydrogenase complex (hydrogenase four or Hyf), a potential formate- and sigma 54-dependent transcriptional activator, HyfR (related to FhlA), and a possible formate transporter, FocB (related to FocA). Five of the nine Hyf-complex subunits are related to subunits of both the E. coli hydrogenase-3 complex (Hyc) and the proton-translocating NADH:quinone oxidoreductases (complex I and Nuo), whereas two Hyf subunits are related solely to NADH:quinone oxidoreductase subunits. The Hyf components include a predicted 523 residue [Ni-Fe] hydrogenase (large subunit) with an N-terminus (residues 1-170) homologous to the 30 kDa or NuoC subunit of complex I. It is proposed that Hyf, in conjunction with formate dehydrogenase H (Fdh-H), forms a hitherto unrecognized respiration-linked proton-translocating formate hydrogenlyase (FHL-2). It is likely that HyfR acts as a formate-dependent regulator of the hyf operon and that FocB provides the Hyf complex with external formate as substrate.