Despite the isolation of an anti-sigma factor over 20 years ago, it is only recently that the concept of an anti-sigma factor emerged as a general mechanism of transcriptional regulation in prokaryotic systems. Anti-sigma factors bind to sigma factors and inhibit their transcriptional activity. Studies on the mechanism of action of anti-sigma factors has shed new light on the regulation of gene expression in bacteria, as the anti-sigma factors add another layer to transcriptional control via negative regulation. Their cellular roles are as diverse as FIgM of Salmonella typhimurium, which can be exported to sense the structural state of the flagellar organelle, to SpoIIAB of Bacillus subtilis participating in the switch from one cell type to another during the process of sporulation. Additionally, the bacteriophage T4 uses an anti-sigma factor to sabotage the Escherichia coli E.sigma 70 RNA polymerase in order to direct exclusive transcription of its own genes. Cross-linking, co-immunoprecipitations, and co-purification indicate that the anti-sigma factors directly interact with their corresponding sigma factor to negatively regulate transcription. In B. subtilis, anti anti-sigma factors regulate anti-sigma factors by preventing an anti-sigma factor from interacting with its cognate sigma factors, thereby allowing transcription to occur.