ABSTRACT Scanning of bacterial genomes to identify essential genes is of biological interest, for understanding the basic functions required for life, and of practical interest, for the identification of novel targets for new antimicrobial therapies. In particular, the lack of efficacious antimicrobial treatments for infections caused by the Burkholderia cepaci a complex is causing high morbidity and mortality of cystic fibrosis patients and of patients with nosocomial infections. Here, we present a method based on delivery of the tightly regulated rhamnose-inducible promoter P _rhaB for identifying essential genes and operons in Burkholderia cenocepacia . We demonstrate that different levels of gene expression can be achieved by using two vectors that deliver P _rhaB at two different distances from the site of insertion. One of these vectors places P _rhaB at the site of transposon insertion, while the other incorporates the enhanced green fluorescent protein gene (e- gfp ) downstream from P _rhaB . This system allows us to identify essential genes and operons in B. cenocepacia and provides a new tool for systematically identifying and functionally characterizing essential genes at the genomic level.