Under resting conditions, steady‐state [Ca] in agonist‐sensitive Ca stores reflects a balance between active uptake (usually mediated by a thap‐sigargin‐sensitive Ca‐ATPase of the SERCA family) and passive efflux of Ca. Even though this pump‐leak cycle appears to be a common property of Ca‐storing organelles, little is known about the nature of the leak pathway. Ca homeostasis in thapsigargin‐sensitive internal Ca stores of single permeabilized BHK‐21 fibroblasts was examined using digital image processing of compartmentalized mag‐fura‐2 (a low‐affinity Ca indicator). It is shown here that the leak of Ca from internal stores is regulated specifically by the cytosolic ATP concentration. The rate of leak was 3.6 times slower in 0.375 mM [ATP] than in 4 mM [ATP] (Na or Mg salt). These effects were observed in the presence of 0 Ca/EGTA, thapsigargin, heparin, and ruthenium red, and therefore appear to be independent of the Ca‐ATPase, the InsP₃ receptor and the ryanodine receptor. The ATP‐stimulated leak was seen in a variety of cell types, including rat basophilic leukemia cells and mouse pancreatic acinar cells. Other nucleotides (ADP, GTP, CTP, and UTP) and nonhydrolyzable ATP analogs (AMP‐PNP and ATPγS) did not reproduce the action of ATP. Changes in cellular metabolism and ensuing alterations in [ATP] will be expected to influence the filling state of internal Ca stores through effects on the passive leak pathway, potentially leading to modulation of Ca signaling and organellar function.—Hofer, A. M., Curci, S., Machen, T. E., Schulz, I. ATP regulates calcium leak from agonist‐sensitive internal calcium stores. FASEB J. 10, 302‐308 (1996)