Hybridization analysis using the Rhizobium meliloti nitrogen fixation gene fixN as a probe revealed the presence of a homologous DNA region in the phytopathogenic bacterium Agrobacterium tumefaciens. Hybridization signals were also detected with total DNAs of Rhizobium leguminosarum bv. phaseoli, Rhodobacter capsulatus and Escherichia coli, but not those of Xanthomonas campestris pv. campestris and Pseudomonas putida. The hybridizing fragment from A. tumefaciens was cloned and sequenced. The predicted gene product of one of the two open reading frames identified on the sequenced fragment shows homology to FixN of different Rhizobiaceae as well as a low but significant similarity to subunit I of heme copper oxidases from various bacteria. The presence of five strictly conserved histidine residues previously implicated in forming ligands to heme and CuB in oxidases and the predicted membrane topology provide evidence that the A. tumefaciens fixN-like gene product is a component of the heme copper oxidase superfamily. The incomplete open reading frame starting only 8 nucleotides downstream of the fixN-like gene exhibits homology to Rhizobium fixO. Using an uidA (GUS) gene fusion it could be shown that the A. tumefaciens fixN-like gene is preferentially expressed under microaerobic conditions. Expression of the uidA fusion is abolished in R. meliloti fixJ and fixK mutants, indicating that an Fnr-like protein is involved in transcriptional regulation of the fixN-like gene in A. tumefaciens. The presence of an upstream DNA sequence motif identical to the Fnr-consensus binding site (anaerobox) further supports this hypothesis. A. tumefaciens mutated in the fixN-like gene shows decreased TMPD-specific oxidase activity under microaerobic conditions, indicating that the fixN-like gene or operon codes for proteins involved in respiration under reduced oxygen availability.