Cytosolic free Ca (Ca(f)) was measured in three different preparations of freshly prepared proximal tubules from the rabbit kidney during energy deprivation using fura-2. Isolated perfused tubules, tubules immobilized on glass cover slips, and tubules in suspension were subjected to inhibitors of oxidative phosphorylation (''chemical hypoxia''); the latter two preparations were also subjected to 40 min of anoxia. During normoxia, Ca(f) ranged from 100 to 180 nM in all three preparations, and chemical hypoxia caused either no change or a small (30-100%) increase in Ca(f) values. Subsequent addition of Ca ionophores increased Ca(f) to 300-500 nM in the first 2 min and to >1 μM after 15 min. In individual experiments, anoxia produced similar responses to those of chemical hypoxia, eliciting no average significant change in Ca(f), despite clear evidence for impaired respiration and plasma membrane damage after 40 min of anoxia. This lack of change in Ca(f) was unrelated to ''Ca buffering'' by fura-2 or inactivation of the dye, since Ca(f) increased to 666 ± 59 nM upon addition of Ca ionophore during anoxia. These data suggest that increased Ca(f) is not a prerequisite for cellular damage during anoxia in proximal renal tubules. Furthermore, no apparent alteration in plasma membrane permeability to Ca occurs before membrane disruption. Decreased ATP seems to initiate a series of Ca(f)-independent events that cause irreversible injury.