Previous genetic analysis of Haemophilus influenzae revealed two mechanisms associated with decreased susceptibility to the novel peptide deformylase inhibitor LBM415: AcrAB-TolC-mediated efflux and Fmt bypass, resulting from mutations in the pump repressor gene acrR and in the fmt gene, respectively. We have isolated an additional mutant, CDS23 (LBM415 MIC, 64 microg/ml versus 4 microg/ml against the parent strain NB65044) that lacks mutations in the acrR or fmt structural genes or in the gene encoding Def, the intracellular target of LBM415. Western immunoblot analysis, two-dimensional gel electrophoresis, and tryptic digestion combined with mass spectrometric identification showed that the Def protein was highly overexpressed in the mutant strain. Consistent with this, real-time reverse transcription-PCR revealed a significant increase in def transcript titer. No mutations were found in the region upstream of def that might account for altered expression; however, pulsed-field gel electrophoresis suggested that a genetic rearrangement of the region containing def had occurred. Using a combination of PCR, sequencing, and Southern blot analyses, it was determined that the def gene had undergone copy number amplification, explaining the high level of target protein expression. Inactivation of the AcrAB-TolC efflux pump in this mutant increased susceptibility 16-fold, highlighting the role of efflux in exacerbating the overall reduced susceptibility resulting from target overexpression.