Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.