Fot the determination of lactose by galactose-oxidase peroxidase (GOP) method, it is necessary to measure the amount of galactose liberated after the hydrolysis of lactose.<BR>(1) Hydrolysis of lactose-Lactose was hydrolyzed with 0.2% H₂SO₄or with living cells of Escherichia coli3-MT (a mutbile-type variant which decomposes lactose but not galactose) . By H₂SO₄method, the calculated dose of galactose was liberated after 2 hours. By MT method, free galactose increased rapidly after 2 hours and reached maximum after 3 hours. The volume of lactose solution was diluted to 1: 4 in the procedures of acid hydrolysis and neutralization, but not in MT method.<BR>(2) Determination of galactose liberated from lactose after hydrolysis- (i) Free galactose in the H₂SO₄hydrolysate of standard solution of lactose was measured by GOP or a cup-plate method using M (galactose-sensitive mutant of enteric bacteria) as the test organism. The amount of galactose was measurable in the range of 25-200γ/2 ml in the case of GOP method, and 1.25-10γ/0.1 ml in the case of M-cup method. (ii) Galactose in the MT hydrolysate was measured by GOP or M-cup method. The amount of galactose could be estimated in the range of 25-100 γ/2 ml by GOP method, but the volume of MT hydrolysate was diluted to 1: 8 because of the necessity of deproteinizing. On the contrary, the amount of 1.25-10γ/0.1 ml could be estimated by M-cup method without the need of deproteinizing.<BR>(3) Determination of lactose in the biological material- (i) Free galactose in the H₂SO₄ hydrolysate of milk was measured by GOP or M-cup method. Although deproteinizing of the hydrolysate was necessary, the determination of galactose in the range of 25-100γ/2 ml was possible by GOP method. By M-cup method, 1.25-10γ/0.1 ml of galactose could be determined without the need of deproteinizing. (ii) The amount of galactose in the MT hydrolysate of milk was measured by GOP or M-cup method. While the estimation of 25-100γ/2 ml of galactose was possible by GOP method after deproteinizing, 1.25-10 γ/0.1 ml of galactose could be estimated by M-cup method without deproteinizing.<BR>From these experimental results, the combination of MT hydrolysis and M-cup method proved to be most sensitive for the determination of lactose in the biological material.