A fluorescent tripeptide, epsilon-N-acetyl-alpha-N-dansyl-L-lysyl-D-alanyl-D-alanine, has been prepared in order to study the thermodynamics and kinetics of the binding of peptides and peptide analogs to the glycopeptide antibiotics. On titration of the tripeptide with typical examples of these antibiotics (vancomycin, ristocetin, alpha- and beta-avoparcin, and teichoplanin), a substantial increase in dansyl fluorescence intensity is observed, allowing the ready determination of binding constants. The binding constants of nonfluorescent ligands can be determined by titration in competition with the fluorescent compound. This new general method for the determination of ligand-binding constants is superior to previous methods in ease of performance, in its sensitivity at low antibiotic concentrations, and in its applicability to ultraviolet-light-absorbing ligands. Biphenomycin gave no indication of binding to D-alanyl-D-alanine-terminating peptides.