During anaerobic growth of Klebsiella pneumoniae on citrate, 9.4 mmol of H₂/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO₂. Upon addition of NiCl₂ (36 µM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H₂-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)⁺ was also achieved by the addition of formate. In crude extracts, the H₂:NAD⁺ oxidoreductase activity was 0.13 µmol min¹ mg¹, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 µmol of NADPH formed min¹ mg¹ at pH 7.0 and 1.7 µmol of NADH formed min¹ mg¹ at pH 9.5. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the Na⁺/H⁺ antiporter monensin, the H₂-dependent reduction of NAD⁺ by membrane vesicles decreased only slightly (about 16%). The NADP⁺- or NAD⁺-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-N-oxide or Triton X-100. NAD(P)H formation with H₂ as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by K. pneumoniae during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H₂:NAD(P)⁺ oxidoreductase that provides reducing equivalents for the synthesis of cell material.