We adapted PCR-based signature-tagged mutagenesis (STM) to Pseudomonas aeruginosa. A collection of 1056 mutants was screened in a chronic lung infection rat model. Thirteen mutants were confirmed to be attenuated. Analysis revealed that these STM mutants represented transposon insertions into eight genes previously described in databases, three genes encoding proteins sharing identity with hypothetical proteins and two genes that shared no significant identity with sequences in databases. Five strains mutated in genes involved in protein degradation, stress tolerance, cation transport, ABC transporter, and an unknown protein were shown to be highly attenuated when tested individually in the rat chronic lung infection model.