Ferredoxins are iron-sulfur electron carrier proteins of sufficiently low redox potentials to transfer electrons to nitrogenase. In Herbaspirillum seropedicae, an endophytic nitrogen-fixing bacterium, the 2[4Fe:4S] ferredoxin gene, fdxA, is located between the clusters nifHDKENXorf1orf2 and nifQmodA1B1C1. In order to elucidate the role of this ferredoxin in nitrogen fixation, the fdxA gene was mutagenized by inserting a lacZ-Km cassette in both orientations. The produced H. seropedicae mutant strains, ALFSAKC (cassette in the same orientation as the gene) and ALFSAKO (opposite orientation), were used for physiological assays. These strains had only 20% of the acetylene-reducing activity compared to the wild type strain SMR1, confirming the importance of FdxA for nitrogen fixation. This assay was also performed under Mo- and/or Fe- limited conditions and the results were essentially the same as those under Mo and Fe sufficiency. Switch off/on experiments revealed that FdxA is not involved in the rapid and reversible nitrogenase inhibition system of H. seropedicae. Expression analysis using the ALFSAKC strain revealed that fdxA is expressed under nitrogen- and oxygen- limiting conditions. A fragment, spanning the region of nifX to the 3' region of nifQ, was cloned into the lacZ promoterless vector pPW452 (pPWnifX-Q) and the b-galactosidase activity levels suggest the absence of a promoter upstream from fdxA, indicating that the nifHDKENXorf1orf2fdxAnifQmodA1B1C1 gene cluster could constitute a single operon. Since the fdxA gene is co-expressed with nif and mod genes, it may be involved in electron transfer to nitrogenase. Supported by CNPq/MCT/Pronex, Fundação Araucária and Paraná Tecnologia