The regulation of aerotaxis in Pseudomonas aeruginosa is reported. P. aeruginosa possesses two aerotaxis transducers, Aer and Aer-2. The aerotactic responses of P. aeruginosa cells were induced during the transition from exponential to stationary growth phase. A deletion mutant for the anaerobic transcriptional regulator ANR showed decreased aerotaxis. The anr mutation eliminated Aer-mediated aerotaxis, but not Aer-2-mediated aerotaxis. Expression of an aer-lacZ transcriptional fusion was also induced during the transition from exponential to stationary growth phase. The anr mutant showed only background levels of aer-lacZ expression. Rapid amplification of cDNA ends (RACE) and DNA sequencing revealed that the 5' end of the mRNA was located at an A nucleotide -67 nt upstream of aer. The aer promoter contained two putative FNR/ANR boxes at -42.5 and -93.5 bp upstream of the transcriptional start site of aer. Mutational analysis of the aer promoter region revealed that both FNR/ANR boxes were essential for the expression of the aer gene. These results indicate that ANR is required for the activation of aer expression but it is not essential for Aer-2-mediated aerotaxis in P. aeruginosa.