σS (RpoS) is the master regulator of the general stress response in Escherichia coli. Several stresses increase cellular σS levels by inhibiting proteolysis of σS, which under non-stress conditions is a highly unstable protein. For this ClpXP-dependent degradation, the response regulator RssB acts as a recognition factor, with RssB affinity for σS being modulated by phosphorylation. Here, we demonstrate that RssB can also act like an anti-sigma factor for σSin vivo, i.e. RssB can inhibit the expression of σS-dependent genes in the presence of high σS levels. This becomes apparent when (i) the cellular RssB/σS ratio is at least somewhat elevated and (ii) proteolysis is reduced (for example in stationary phase) or eliminated (for example in a clpP mutant). Two modes of inhibition of σS by RssB can be distinguished. The ‘catalytic’ mode is observed in stationary phase cells with a substoichiometric RssB/σS ratio, requires ClpP and therefore probably corresponds to sequestering of σS to Clp protease (even though σS is not degraded). The ‘stoichiometric’ mode occurs in clpP mutant cells upon overproduction of RssB to levels that are equal to those of σS, and therefore probably involves binary complex formation between RssB and σS. We also show that, under standard laboratory conditions, the cellular level of RssB is more than 20-fold lower than that of σS and is not significantly controlled by stresses that upregulate σS. We therefore propose that antisigma factor activity of RssB may play a role under not yet identified growth conditions (which may result in RssB induction), or that RssB is a former antisigma factor that during evolution was recruited to serve as a recognition factor for proteolysis.