During carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30–50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation- induced csi::lacZ fusions using the λplac Mu system. One operon fusion (csi 2::lacZ) has been studied in detail. csi 2::lacZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi 2::lacZ did not synthesize at least 16 proteins present In an isogenic csi 2+ strain. Cells containing csi 2::lacZ or csi 2::Tn10 did not produce glycogen, did not develop thermo-tolerance and H2O2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::lacZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi 2::lacZ defines a regulatory gene of central importance for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi 2 demonstrated that the csi 2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi 2/katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose ‘rpoS’ (‘σs’) as appropriate designations.