There has been considerable debate as to whether adenosine triphosphate (ATP) is compartmentalized within cells and, in particular, whether the ATP concentration directly beneath the plasma membrane, experienced by membrane proteins, is the same as that of the bulk cytoplasm. This issue has been difficult to address because there is no indicator of cytosolic ATP, such as those available for Ca2+, capable of resolving the submembrane ATP concentration ([ATP](sm)) in real time within a single cell. We show here that mutant ATP-sensitive K+ channels can be used to measure [ATP](sm) by comparing the increase in current amplitude on patch excision with the ATP dose-response curve. In Xenopus oocytes, [ATP](sm) was 4.6 ± 0.3 mM (n = 29) under resting conditions, slightly higher than that measured for the bulk cytoplasm (2.3 mM). In mammalian (COSm6) cells, [ATP](sm) was slightly lower and averaged 1.4 ± 0.1 mM (n = 66). Metabolic poisoning (10 min of 3 mM azide) produced a significant fall in [ATP](sm) in both types of cells: to 1.2 ± 0.1 mM (n = 24) in oocytes and 0.8 ± 0.11 mM for COSm6 cells. We conclude that [ATP](sm) lies in the low millimolar range and that there is no gradient between bulk cytosolic and submembrane [ATP].