From 2000–2001, over 10 studies have been completed, using signature-tagged mutagenesis, on a variety of bacterial pathogens. Investigators are examining the attenuated strains in additional in vivo, cell culture or in vitro assays to further characterize and catalog the mutants. Notable advances included screening of the attenuated mutants en masse in other assays, verification that mutant strains are defective for growth in specific host tissues, and identification of potential live-attenuated vaccine candidates. In addition, researchers are using signature-tagged strains to characterize the infection process, which is providing snapshots of bottlenecks and quantitative measurements of bacterial spread from tissue to tissue. This review focuses on themes emerging from signature-tagged mutagenesis studies completed on bacterial pathogens since mid-2000.