s S (RpoS), the master regulator of the general stress response in Escherichia coli, is a model system for regulated proteolysis in bacteria. s S turnover requires ClpXP and the response regulator RssB, whose phosphorylated form exhibits high af®nity for s S . Here, we demonstrate that recognition by the RssB/ClpXP system involves two distinct regions in s S . Region 2.5 of s S (a long a-helix) is suf®cient for binding of phosphorylated RssB. However, this interaction alone is not suf®cient to trigger proteolysis. A second region located in the N-terminal part of s S , which is exposed only upon RssB±s S interaction, serves as a binding site for the ClpX chaperone. Binding of the ClpX hexameric ring to s S -derived reporter proteins carrying the ClpX-binding site (but not the RssB-binding site) is also not suf®cient to commit the protein to degradation. Our data indicate that RssB plays a second role in the initiation of s S proteolysis that goes beyond targeting of s S to ClpX, and suggest a model for the sequence of events in the initiation of s S proteolysis.