The structural genes (hupSL) of the membrane-bound NiFe-containing H2-uptake hydrogenase (Hup) of Azotobacter chroococcum were identified by oligonucleotide screening and sequenced. The small subunit gene (hupS) encodes a signal sequence of 34 amino acids followed by a 310-amino-acid, 34156D protein containing 12 cysteine residues. The large subunit gene (hupL) overlaps hupS by one base and codes for a predicted 601-amino-acid, 66433D protein. There are two regions of strong homology with other Ni hydrogenases: a Cys-Thr-Cys-Cys-Ser motif near the N-terminus of HupS and an Asp-Pro-Cys-Leu-Ala-Cys motif near the carboxy-terminus of HupL. Strong overall homology exists between Azotobacter, Bradyrhizobium japonicum and Rhodobacter capsulatus Hup proteins but less exists between the Azotobacter proteins and hydrogenases from Desulfovibrio strains. Mutagenesis of either hupS or hupL genes of A. chroococcum yielded Hup- phenotypes but some of these mutants retained a partial H2-evolving activity. Hybridization experiments at different stages of gene segregation confirmed the multicopy nature of the Azotobacter genome.