Biofilms are adherent aggregates of bacterial cells that form on biotic and abiotic surfaces, including human tissues. Biofilms resist antibiotic treatment and contribute to bacterial persistence in chronic infections. Hence, the elucidation of the mechanisms by which biofilms are formed may assist in the treatment of chronic infections, such as Pseudomonas aeruginosa in the airways of patients with cystic fibrosis. Here we show that subinhibitory concentrations of aminoglycoside antibiotics induce biofilm formation in P. aeruginosa and Escherichia coli. In P. aeruginosa, a gene, which we designated aminoglycoside response regulator (arr), was essential for this induction and contributed to biofilm-specific aminoglycoside resistance. The arr gene is predicted to encode an inner-membrane phosphodiesterase whose substrate is cyclic di-guanosine monophosphate (c-di-GMP)-a bacterial second messenger that regulates cell surface adhesiveness. We found that membranes from arr mutants had diminished c-di-GMP phosphodiesterase activity, and P. aeruginosa cells with a mutation changing a predicted catalytic residue of Arr were defective in their biofilm response to tobramycin. Furthermore, tobramycin-inducible biofilm formation was inhibited by exogenous GTP, which is known to inhibit c-di-GMP phosphodiesterase activity. Our results demonstrate that biofilm formation can be a specific, defensive reaction to the presence of antibiotics, and indicate that the molecular basis of this response includes alterations in the level of c-di-GMP.