Pseudomonas aeruginosa harbors a chromosomal aminoglycoside phosphotransferase gene, aph(3')-IIb, which confers P. aeruginosa resistance to several important aminoglycoside antibiotics, including kanamycin A and B, neomycin B and C, butirosin, and seldomycin F5. The aph(3')-IIb gene has been found to be regulated by an AraC-type transcriptional regulator (HpaA) encoded by a gene located upstream of the aph(3')-IIb gene. In the presence of 4-hydroxyphenylacetic acid (4-HPA), HpaA activates the expression of aph(3')-IIb as well as that of the hpa regulon which encodes metabolic enzymes for the utilization of 4-HPA. hpaA and aph(3')-IIb form an operon, and in response to the presence of 4-HPA, the wild-type P. aeruginosa strain PAK (but not its hpaA mutant strain) displays increased resistance to neomycin. A survey of 39 clinical and 19 environmental isolates of P. aeruginosa demonstrated in all of them the presence of an hpaA-aph gene cluster, while 56 out of the 58 isolates are able to utilize the 4-HPA as a sole carbon source, suggesting a feature common to P. aeruginosa strains. Interestingly, a larger portion of clinical isolates than environmental isolates showed 4-HPA-induced resistance to neomycin. The aph(3')-IIb gene product is likely to function as a metabolic enzyme which has a cross-reactivity with aminoglycosides. These findings provide new insight into the possible mechanism of P. aeruginosa antibiotic resistance.