The nucleotide sequence of a 2.5-kbp region following the hydrogenase structural genes (hupSL) in the H2 uptake gene cluster from Rhizobium leguminosarum bv. viciae UPM791 was determined. Four closely linked genes encoding peptides of 27.9 (hupC), 22.1 (hupD), 19.0 (hupE), and 10.4 (hupF) kDa were identified immediately downstream of hupL. Proteins with comparable apparent molecular weights were detected by heterologous expression of these genes in Escherichia coli. The six genes, hupS to hupF, are arranged as an operon, and by mutant complementation analysis, it was shown that genes hupSLCD are cotranscribed. A transcription start site preceded by the -12 to -24 consensus sequence characteristic of NtrA-dependent promoters was identified upstream of hupS. On the basis of the lack of oxygen-dependent H2 uptake activity of a hupC::Tn5 mutant and on structural characteristics of the protein, we postulate that HupC is a b-type cytochrome involved in electron transfer from hydrogenase to oxygen. The product from hupE, which is needed for full hydrogenase activity, exhibited characteristics typical of a membrane protein. The features of HupC and HupE suggest that they form, together with the hydrogenase itself, a membrane-bound protein complex involved in hydrogen oxidation.