In the displacement study, the effects of tolbutamide on the absorption spectra and induced CD spectra of 2-(4'-hydroxyphenylazo)benzoic acid(HABA)-bovine serum albumin (BSA) complex were investigated using the samples obtained from equilibrium dialysis at pH 7.40. The binding of tolbutamide to BSA enhanced the binding of the azo form in spite of the displacement of the hydrazone form of bound HABA at the molar ratio of HABA to BSA of 0.65, causing the increase in the negative ellipticity at 445 nm, which was termed the C band. The C band could be assigned to the azo form of bound HABA.