The nucleotide sequence was obtained for the hypE gene in the cluster of structural and accessory genes required for the assembly and functioning of the membrane-bound, dimeric, (NiFe)hydrogenase in Azotobacter vinelandii. The hypE gene encodes a polypeptide of 341 amino acid residues which is rich in alanine, glycine, valine and proline and appears to be involved in maturation of the enzyme because chromosomal mutations in hypE block O2-dependent H2-oxidation and affect the amount, processing and localization of the (NiFe) hydrogenase alpha-subunit. The complete nucleotide sequence for the hydrogenase gene cluster in A. vinelandii has now been assembled into a contiguous sequence of 13,914 bp containing 16 potential genes which appear to be transcribed undirectionally. They are arranged in the order hoxK, hoxG, hoxZ, hoxM, hoxL, hoxO, hoxQ, hoxR, hoxT, hoxV, hypA, hypB, hypF, hypC, hypD and hypE. This cluster closely resembles those described for comparable (NiFe) hydrogenases in other bacteria.