The effects of leaf disc size, extraction solvents and the time of heating and cooling on the ATP extraction from the green leaves as well as the optimal MgSO₄ concentration and pH on the luciferin-luciferase enzyme reaction system with ATP were investigated. Leaf discs were prepared by using the leaf disc-punch from the fully expanded top-leaf blade of rice (Oryza sativa L.) and soybean (Glycine max Merr, ) plants grown in the glass house or in the field in pot. These discs with buffer solution or distilled water (D.W.) were dipped in the boiling water for several minutes and then cooled for several hours with mild shaking under the cold condition (3°C) to extract ATP from leaves as a disc. ATP in the extract was determined from the luminescence intensity induced by the enzyme reaction between ATP in the extract and luciferin-luciferase in the FLE-50 (Sigma Chem. Co.). Luminescence intensity was measured by ATP photometer (Type 2000, SAI Tech. Co. USA). Following results were obtained: 1. In this experiment, 15 ml of 13.33 mM MgSO₄ were added to FLE-50, which contained 6.67 mM MgSO₄ as a kit, to give 20 mM MgSO₄ as a optimal concentration for enzyme reaction (Fig. 1). 2. Luciferin-luciferase reaction with ATP was inhibited by potassium-phosphate buffer and CaCl₂, but not by HEPES- and Tris-buffer and D. W. (Table 1). Then, 50 mM HEPES buffer (pH 7.2), which optimal pH of this enzyme reaction was 7.2-7.4 as same as explained in the FLE-50 kit, was used as the buffer of extraction and determination of ATP in the leaf discs. 3. The effective extraction of ATP from the discs by the boiling water extraction method was obtained by using the discs with 2.0-3.0mm diameter in size with HEPES buffer (pH 7.2, 5 pieces/2.5 ml), heating them for 0.5-1.0 min in the boiling water and subsequent cooling for about 5 hr at 3°C (Fig. 2 and Fig. 3). 4. The boiling water extraction method was compared with the cold perchlorate extraction. The data obtained by the boiling water method were consistently higher than by perchlorate extraction method (Table 3). 5. Each variety in rice and soybean plants has different ATP contents within the limits of experiment. The ranking of these varietal differences in each plant did not change between experiments except Shin No. 2, one of soybean variety (Table 4). 6. There were no clear correlationships between ATP and chlorophyll contents in each plant (Fig. 4). These results mentioned above were discussed with reference to the availability of the boiling water extraction method for ATP extraction from green leaves.