LY191145 is an N-alkylated glycopeptide antibiotic (the p-chlorobenzyl derivative of LY264826) with activity against vancomycin-susceptible and -resistant bacteria. Similar to vancomycin, LY191145 inhibited polymerization of peptidoglycan when muramyl pentapeptide served as a substrate but not when muramyl tetrapeptide was used, signifying a substrate-dependent mechanism of inhibition. Examination of ligand binding affinities for LY191145 and the effects of this agent on R39 D,D-carboxypeptidase action showed that, similar to vancomycin, LY191145 had an 800-fold greater affinity for N,N'-diacetyl-L-Lys-D-Ala-D-Ala than for N,N'-diacetyl-L-Lys-D-Ala-D-Lac. The antibacterial activity of LY191145 was antagonized by N,N'-diacetyl-L-Lys-D-Ala-D-Ala, but the molar excess required for complete suppression exceeded that needed to suppress inhibition by vancomycin. LY191145 is strongly dimerized and the p-chlorobenzyl side chain facilitates interactions with bacterial membranes. These findings are consistent with a mechanism of inhibition where interactions between antibiotic and D-Ala-D-Ala or D-Ala-D-Lac residues depend on intramolecular effects occurring at the subcellular target site.