The ATP-dependent Clp protease of Escherichia coli consists of two subunits, the ClpP subunit, which has the proteolytic active site, and ClpA, which possesses ATPase activity and activates the proteolytic activity of ClpP in vitro. Recently, Zylicz and co-workers (Wojtkowiak, D., Georgopoulos, C., and Zylicz, M. (1993) J. Biol. Chem. 268, 22609-22617) identified another E. coli protein that activated ATP-dependent degradation of lambda O protein in the presence of ClpP. The amino-terminal sequence of this protein corresponds to the translated amino-terminal sequence of a gene that we have named clpX. clpX encodes a protein with M(r) 46,300, similar to that observed for the protein purified by Wojtkowiak et al. clpX is an operon with clpP; both genes are cotranscribed in a single heat-inducible 2200-base mRNA, with clpP the promoter proximal gene. The sequence of ClpX includes a single consensus ATP-binding site motif and has limited homology to regions of ClpA and other members of the ClpA/B/C family. A third group of proteins, ClpY, closely related to ClpX, has been identified by sequence homology. Mutations in either clpX or clpP abolish degradation of the highly unstable lambda O protein in vivo. clpX mutants are not defective in degradation of previously identified ClpA/ClpP substrates such as a ClpA-beta-galactosidase fusion protein. It appears that selectivity of degradation by ClpP in vivo is determined by interaction of ClpP with different regulatory ATPase subunits.