Previously, the deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the Bradyrhizobium japonicum hydrogenase structural genes caused lack of normal-sized hydrogenase (Hup) subunits and complete loss of Hup activity. It was suggested that this region encodes one or more genes required for Hup processing. Sequencing of a 3322-bp XcmI fragment of DNA covering this 2.9-kb EcoRI fragment within the hup gene cluster revealed the presence of five open reading frames (ORFs) designated hupG, hupH, hupI, hupJ and hupK, encoding polypeptides with calculated molecular masses of 15.8, 30.7, 7.6, 18.1 and 38 kDa, respectively. Based on deduced amino acid (aa) sequences, all five products of the hupGHIJK genes showed significant homology with other genes' products in several H2-utilizing bacteria. Of particular interest are HupG and HupI. HupG showed 70% similarity (28% identity) to the HyaE of the Escherichia coli hydrogenase-1 operon which was demonstrated to be involved in the processing of hydrogenase-1. HupI showed strong identity to rubredoxin and rubredoxin-like proteins from many other bacteria. The latter proteins contain two 'C-X-X-C' motifs, which may serve as iron ligands for non-heme iron proteins involved as intermediate electron carriers or in the assembly process for Fe-S (or NiFe-S) clusters.