Bovine k-casein macropeptide was prepared by adding TCA (3, 6, and ) treatment after chymosin reaction. Each TCA soluble macropeptide was fractionated into five peak by ion exchange column chromatography. In proportiion to TCA concentrations, the ratio of peak area showed different the elution pattern. At the 6 and TCA concentration, area ratio of P-I which did not content carbohydrates was decreased to 19.9 and of total peak area respectively. The area of P-III was changed from when the TCA concentration was increased from 3 to 6 and Cholera toxin binding activity of k-casein macropeptide eluted at NaCl gradient was not inhibited by 6 and TCA treatments. The use of TCA as extraction buffer was feasible and led to an effective separation of the peak III.