Nickel is a component of the H2-oxidizing hydrogenase of many bacteria. We report that nickel is required not only for the activity of the Bradyrhizobium japonicum H2 uptake (hup) enzyme but also for the initiation of its transcription. A much greater level of hydrogenase-specific mRNA was detected in cells that were derepressed for hydrogenase in the presence of 5 microM nickel than in the absence of nickel. Control experiments involving probing of mRNA with a B. japonicum gene encoding a non-nickel-containing protein (delta-aminolevulinic acid synthetase) demonstrated that there was no influence by nickel levels on its message. Assays utilizing a plasmid-borne gene fusion linking the 5' upstream sequence of the hup locus to a promoterless beta-Gal structural gene demonstrated that an upstream region between -239 and -168 is critical for transcriptional regulation by nickel. Hydrogenase transcription is not self-regulated by the nickel-containing hydrogenase as the hydrogenase promoter was still regulated by nickel in a mutant strain containing a Tn5 insertion in the hup structural gene. This is the first report of transcriptional regulation of a protein by nickel.