The binding of doxycycline to HSA under simulated physiological conditions (pH 7.4, 67 rnM phosphate, I = 0.17, drug concentration 100 mu M, HSA concentration up to 475 mu M, 36.5 degrees C) was studied by CE-frontal analysis. The number of primary binding sites, binding constant and physiological protein-binding percentage were 1.9, 1.51 X 10(3)M(-1) and 59.80%, respectively. In addition, the thermodynamic parameters including enthalpy change (Delta H) entropy change (Delta S) and free energy change (Delta G) of the reaction were obtained in order to characterize the acting forces between doxycycline and HSA. Furthermore, to better understand the nature of doxycycline-HSA binding and to get information about potential interaction with other drugs, displacement experiments were performed. The results showed that doxycycline binds at site II of HSA.