The accurate classification of leukemia is very important, because antileukemic agents have different effects for each leukemic types. But still now, it is often difficult, especially in acute leukemias. The urine hydrolysis test was first proposed by Brachet and then Thoma and Gardner et al, who suggested that nuclear chromatin of myelocytic cells were "hydrolysed", while lymphocytic and monocytic nuclei remained intact. The purpose of this study were to establish the most considerable conditions to carry out this test, and to confirm the very responsible substance in urine for this test. Certainly, the lytic action of the urine on nuclear chromatin were variable in different conditions. Therefore, 3 factors, pH and temperature of the urine and incubation time were discussed for the purpose of standardization of Brachet's test. The temperature of urine specimen was heated at 60℃ in preliminary studies. But it was demonstrated that the most effective temperature of this test was 37℃ from aspects of its biochemical natures in this paper, so I tried to design the best method. It was the best method with temperature of 37℃ with incubation time of 10, 20, 30, and 40 minutes for each blood film, which showed satisfactory results in hydrolyze myelocytic cell nuclear chromatin to differentiate from other cells. The optimum pH range of the urine for this reaction were different in blood film and in bone-marrow film. Nevertheless, it was noted that the range of pH 6.2 to 6.8 was common and suitable for both films. The urine substance responsible for this test was studied further. The effect of varying concentration of salt solution and pure distilled water were tried in same method instead of the urine with various conditions but they showed only diffused nonspecific effect, not like the urine specimen. Also purified enzyme solution of RNase in buffered solution and in distilled water resulted nonspecific reaction. On the other hand, the reaction of crystaline DNase solution applied to blood film were markedly simi1ar to those of the Brachet's test. The DNase activities on the urine, were parallel very well with the results of the Brachet's test. In addition, the activities of the Brachet's test fell down linialy with dilution of the urine by buffered solution. From these, the hydrolysis of nuclear chromatin of cells seemed to be due to DNase of urine most likely. Especially, DNase I must be responsible for the Brachet's test because the optimum pH and temperature in this test were at pH 6.6 to 6.8 at 37℃, while no activity was seen at pH 5.0. And this test was inhibited by citrate and not by MgSo4. In clinical application, it was observed that this Brachet's test was exactly useful in differentiation of the type of leukemic patients.