Cellular ATP is commonly determined as production of bioluminescence using a luciferin-luciferase reaction system. Before the measurement of bioluminescence, cellular ATP must first be extracted. Two commonly used extraction methods are: () Tris-borate buffer (pH 9.2) coupled with a heating process (to inactivate ATPase) and () perchloric acid followed by neutralization. However, we found that both Tris-borate buffer and perchloric acid interfered with the luciferin-luciferase system. Here, we report a convenient single-step boiling deionized water (DW) method for extracting cellular ATP to replace perchloric acid and Tris-borate buffer. We showed that the boiling DW method did not interfere with the bioluminescence and was effective in inhibiting ATPase. This improved method required no neutralization and dilution and thus was more convenient than the perchloric acid method. Unlike the Tris-borate/heating procedure, our method did not require a separate heating step because boiling DW effectively inhibited ATPase and thus accomplished the two missions in one step for both suspended and attached cells. The improved method was precise for both suspended cells and attached cells, when cell numbers were between 10(3) and 10(6). The method also was more sensitive than other methods because it required much fewer cells (10(4) to 10(5)) than other methods for ATP determination. Thus, this one-step method is suitable for routine assay of cellular ATP for both suspended and attached cells.