Previous studies have shown that cytosolic-free Ca 2 ϩ (Ca f ) increases to at least low micromolar concentrations during ATP depletion of isolated kidney proximal tubules. However, peak levels could not be determined precisely with the Ca 2 ϩ -sensitive fluorophore, fura-2, because of its high affinity for Ca 2 ϩ . Now, we have used two low affinity Ca 2 ϩ fluorophores, mag-fura-2 (furaptra) and fura-2FF, to quantitate the full magnitude of Ca f increase. Between 30 and 60 min after treatment with antimycin to deplete ATP in the presence of glycine to prevent lytic plasma membrane damage, Ca f measured with mag-fura-2 exceeded 10 M in 91% of tubules studied and 68% had increases to greater than 100 M. Ca f increases of similar magnitude that were dependent on influx of medium Ca 2 ϩ were also seen using the new low Ca 2 ϩ affinity, Mg 2 ϩ -insensitive, fluorophore fura-2FF in tubules depleted of ATP by hypoxia, and these increases were reversed by reoxygenation. Total cell Ca 2 ϩ levels in antimycin-treated or hypoxic tubules did not change, suggesting that mitochondria were not buffering the increased Ca f during ATP depletion. Considered in the context of the high degree of structural preservation of glycine-treated tubule cells during ATP depletion and the commonly assumed Ca 2 ϩ requirements for phospholipid hydrolysis, actin disassembly, and Ca 2 ϩ -mediated structural damage, the remarkable elevations of Ca f demonstrated here suggest an unexpected resistance to the deleterious effects of increased Ca f during energy deprivation in the presence of glycine. ( J.