The putative products of six Azotobacter vinelandii chromosomal open reading frames (ORFs) were suggested to be involved in dihydrogen (H-2) metabolism [Chen and Mortenson (1992) Biochim Biophys Acta 1131, 199-202]. A promoterless lacZ-containing cassette was used to disrupt the ORFs. Qualitative analysis revealed that the lacZ genes were expressed only in those mutants where the directions of the inserted lacZ were identical to those of the ORFs, showing that the six ORFs were transcribed as predicted. Unlike wildtype (w.t.), none of the mutants could perform dioxygen (O-2)-dependent Hz-oxidation, even though Western immunoanalyses showed that the hydrogenase large subunit was present although in amounts less than in w.t. Only one of the mutants (a hypB mutant), grown in nickel-enriched media, showed meaningful restoration of the H-2-oxidizing ability. From the above observations it is concluded that (a) the six-ORF region is transcriptionally active and involved in H-2-oxidation, (b) the product of hypB is needed for nickel activation of hydrogenase, and (c) the six ORFs (genes) belong to two or more operons. Possible roles of the gene products for the assembly, modification, and processing of hydrogenase from its apoproteins and metal centers are discussed.