A gene library of chromosomal DNA from Pseudomonas aeruginosa contained a DNA fragment which was able to restore anaerobic growth to an Escherichia coli fnr deletion mutant on glycerol/nitrate medium. The cloned gene (termed anr) was sequenced and shown to encode a protein of 244 amino acids with a calculated molecular weight of 27,129. The deduced amino acid sequence of the anr gene product showed considerable similarity to the FNR protein from E. coli. Expression of the anr gene in a T7 promoter/polymerase system identified ANR as a 31 kDa protein. Transcriptional analysis of the anr gene showed that it is monocistronic but apparently lacks the equivalent sites for negative autoregulation which have been shown to be present in the promoter region of the E. coli fnr gene. The ANR protein was shown to activate transcription of the pfl gene in E. coli in response to anaerobiosis, as well as being able to restore the activity of three anaerobically inducible enzymes. A P. aeruginosa mutant incapable of growing anaerobically with nitrate or on arginine was fully complemented by the anr gene, indicating that it probably has a function in controlling anaerobic gene expression in Pseudomonas. Further corroboration for this assumption was provided by S1 nuclease analysis of transcription of the multiple promoters of the E. coli pfl operon in P. aeruginosa. Transcription was induced by oxygen limitation and was completely ANR-dependent in both aerobic and anaerobic cells. Removal of the upstream regulatory sequence of the pfl operon, which includes the sequences required for FNR-dependent regulation in E. coli, removed ANR-dependent transcriptional control of the remaining pfl promoters, irrespective of the cellular oxygen status. These results imply that the mechanisms by which ANR and FNR regulate transcription are fundamentally similar.