The Escherichia coli protease HtrA has two PDZ domains, and sequence alignments predict that the E. coli protease Tsp has a single PDZ domain. PDZ domains are composed of short sequences (80-100 amino acids) that have been implicated in a range of protein:protein interactions. The PDZ-like domain of Tsp may be involved in binding to the extreme COOH-terminal sequence of its substrate, whereas the HtrA PDZ domains are involved in subunit assembly and are predicted to be responsible for substrate binding and subsequent translocation into the active site. E. coli has a system of protein quality control surveillance mediated by the ssrA-encoded peptide tagging system. This system tags misfolded proteins or protein fragments with an 11-amino acid peptide that is recognized by a battery of cytoplasmic and periplasmic proteases as a degradation signal. Here we show that both HtrA and Tsp are able to recognize the ssrA-encoded peptide tag with apparent K(D) values of approximately 5 and 390 nm, respectively, and that their PDZ-like domains mediate this recognition. Fusion of the ssrA-encoded peptide tag to the COOH terminus of a heterologous protein (glutathione S-transferase) renders it sensitive to digestion by Tsp but not HtrA. These observations support the prediction that the HtrA PDZ domains facilitate substrate binding and the differential proteolytic responses of HtrA and Tsp to SsrA-tagged glutathione S-transferase are interpreted in terms of the structure of HtrA.