In growing Escherichia coli cells, the master regulator of the general stress response, σS (RpoS), is subject to rapid proteolysis. In response to stresses such as sudden carbon starvation, osmotic upshift or shift to acidic pH, σS degradation is inhibited, σS accumulates and numerous σS-dependent genes with stress-protective functions are activated. σS proteolysis is dependent on ClpXP protease and the response regulator RssB, whose phosphorylated form binds directly to σSin vitro. Here, we show that substitutions of aspartate 58 (D58) in RssB, which result in higher σS levels in vivo, produce RssB variants unable to bind σSin vitro. Thus, RssB is the direct substrate recognition factor in σS proteolysis, whose affinity for σS depends on phosphorylation of its D58 residue. RssB does not dimerize or oligomerize upon this phosphorylation and σS binding, and RssB and σS exhibit a 1:1 stoichiometry in the complex. The receiver as well as the output domain of RssB are required for σS binding (as shown in vivo and in vitro) and for complementation of an rssB null mutation. Thus, the N-terminal receiver domain plays an active and positive role in RssB function. Finally, we demonstrate that RssB is not co-degraded with σS, i.e. RssB has a catalytic role in the initiation of σS turnover. A model is presented that integrates the details of RssB–σS interaction, the RssB catalytic cycle and potential stress signal input in the control of σS proteolysis.