Hydrogenase defective mutants of Escherichia coli HB101 were isolated by MNNG-mutagenesis and methyl viologen filter paper screening method. E. coli DNA was digested with restriction endonuclease EcoRI and ligated into the EcoRI site of plasmid pBR322. The resulting recombinant plasmids were used to transform E. coli HY1, a mutant with a defective hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for a E. coli hydrogenase in plasmid pHY1-10. This plasmid was not able to complement the other mutants, E. coli HY2, HY3, and LCB850. Polyacrylamide gel electrophoresis was used to analyze the hydrogenase(s) of the wild type, mutants, and mutant strains transformed with pHY1-10.