Derepression of an uptake hydrogenase in Bradyrhizobium japonicum is dependent on a microaerophilic environment. Addition of DNA gyrase inhibitors during depression of hydrogenase specifically prevented expression of the hydrogenase enzyme. Antibodies to individual hydrogenase subunits failed to detect the protein after derepression in the presence of inhibitors, although there was no general inhibition of protein synthesis. The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was not significantly different whether proteins were labeled in the presence or in the absence of gyrase inhibitors. In contrast, if transcription or translation was inhibited by addition of inhibitors of those functions, virtually no proteins were labeled during derepression. This indicated that most of the 14C-labeled proteins were synthesized de novo during derepression, synthesis of most proteins was unaffected by gyrase inhibitors, and the dependence of hydrogenase synthesis on gyrase activity was a specific one.