High molecular mass penicillin-binding proteins (PBPs, DD-peptidases) of class B, such as Streptococcus pneumoniae PBP2x, catalyze the cross-linking of peptidoglycan in bacterial cell wall biosynthesis and are thus important antibiotic targets. Despite their importance in this regard, structure-function studies of ligands of these enzymes have been impeded by the absence of useful substrates. In vitro, these enzymes do not catalyze peptide hydrolysis or aminolysis, their in vivo reaction, but some, such as PBP2x, do catalyze these reactions of certain thioesters such as PhCH2CONHCH2COSCH(D-Me)CO2- (2). We have now prepared several peptidoglycan-mimetic thioesters that we expected to more closely resemble the natural substrates of these enzymes. To our surprise, however, these compounds, although indeed substrates of PBP2x, did not, unlike 2, appear to form an acyl-enzyme intermediate during hydrolysis, and their turnover was inhibited by certain peptides and N-acylamino acids much more weakly than that of 2. An inhibitor of this type, N-benzyloxycarbonyl-d-glutamic acid, also quenched the fluorescence of PBP2x that had been labeled at the DD-peptidase active site by 6-dansylamidopenicillanic acid. These results were interpreted in terms of a model where the peptidoglycan-mimetic thioesters preferentially bound to and hydrolyzed at a site other than the classical DD-peptidase active site. This second site is likely to represent part of an extended binding site that accommodates a peptidoglycan substrate or regulator in vivo. Such a site may be a target for future inhibitor/antibiotic design.