This work examines the electrostatic interactions involved in the recognition of D-Ala-D-Ala (DADA) by vancomycin (Van) by using capillary electrophoresis (CE) and affinity capillary electrophoresis (ACE). Acetylation of the N-terminal amine of Van decreases its affinity for Di-Ac-L-Lys-D-Ala-D-Ala (Ac(2)KDADA) by a factor of 11 at pH 7.1 (from 4.3 mu M to 48 mu M). Succinylation of the N-terminus of Van introduces a pendant negative charge that further decreases its affinity for Ac(2)KDADA about 2-fold at pH 7.1. The association of Ac-D-Ala-D-Ala (Ac-D-Ala) with Van shifts the pK(a) of the N-terminal amine of Van by 1.7 units from 7.1 to 8.8, and thus changes its net charge in the range of values of pH between 6 and 10. The electrostatic interaction between the -CO2- group of the DADA moiety and the -NH2+CH3 group of Van contributes approximately 5.9 kJ/mol to the free energy of binding of these species. In addition to establishing or confirming these thermodynamic parameters, this paper illustrates the use of CE as a physical-organic tool in examining electrostatic interactions in biomolecular recognition.