Abstract: The interaction of tetracycline hydrochloride to Bovine Serum Albumin (BSA) at various temperatures and pH values using Equilibrium Dialysis (ED) method was studied. Scatchard analysis of the binding data revealed the presence of one high affinity binding site with k1 value of 1.67x106 M-1 and six low affinity binding sites with k2 value of 1.44x105 M-1 at pH 7.4 and 25°C. Site-specific probe displacement data suggested that warfarin site (site-I) is the high affinity binding site and benzodiazepine site (site-II) is the low affinity binding site on BSA for this drug. The high affinity binding site was found to be affected by temperature and pH of the medium. The thermodynamic data indicated that the binding process of tetracycline hydrochloride to BSA is spontaneous, exothermic and entropically driven. Electrostatic forces, hydrogen bonding, hydrophobic interactions and van der Waals forces are probably involved in the overall binding process of tetracycline hydrochloride to BSA. The affinity of this drug to BSA is dependent on the conformational changes of BSA caused by N-B transition.