Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised patients and those with cystic fibrosis genetic disease. To identify new virulence genes of P. aeruginosa, a selection system was developed based on the in vivo expression technology (IVET) that was first reported in Salmonella system. An adenine-requiring auxotrophic mutant strain of P. aeruginosa was isolated and found avirulent on neutropenic mice. A DNA fragment that can complement the mutant strain, containing purEK operon that is required for de novo biosynthesis of purine, was sequenced and used in the IVET vector construction. By applying the IVET selection system to a neutropenic mouse infection model, genetic loci that are specifically induced in vivo were identified. Twenty-two such loci were partially sequenced and analyzed. One of them was a well-studied virulence factor, pyochelin receptor (FptA), that is involved in iron acquisition. Fifteen showed significant homology to reported sequences in GenBank, while the remaining six did not. One locus, designated np20, encodes an open reading frame that shares amino acid sequence homology to transcriptional regulators, especially to the ferric uptake regulator (Fur) proteins of other bacteria. An insertional np20 null mutant strain of P. aeruginosa did not show a growth defect on laboratory media; however, its virulence on neutropenic mice was significantly reduced compared with that of a wild-type parent strain, demonstrating the importance of the np20 locus in the bacterial virulence. The successful isolation of genetic loci that affect bacterial virulence demonstrates the utility of the IVET system in identification of new virulence genes of P. aeruginosa.