This study tests the hypothesis that intracellular {dollar}pH\ (pH\sb{lcub}i{rcub}){dollar} and ionized cytosolic calcium ( (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar}) deregulation relate to reversible and irreversible responses to anoxia with and without inhibition of glycolysis. Effects of anoxia on (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} were studied in cultured proximal tubular epithelial cells (PTE) correlated with mitochondrial function and cell viability. After 5 hours of exposure to oxygen-depleted medium, ATP slowly declined by 30% associated with minimal cell killing. When iodoacetic acid was added to the argon-bubbled perfusate, ATP decreased precipitously and was not measurable by 30 min. This was associated with 80% LDH release by 3 hr. Changes in (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} exhibited three distinct phases. Phase 1 showed no change in (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} for a variable time, was shortened dramatically by inhibition of glycolysis, and produced a rapid and uniform (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} elevation after 30 minutes of hypoxia. Phase 2 was characterized by a rapid increase in (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar}. Phase 3 was characterized by buffering of Ca{dollar}\sp{lcub}2+{rcub}{dollar}-reflecting intrinsic buffering mechanisms in Ca{dollar}\sp{lcub}2+{rcub}{dollar}-overloaded cells. The rapid drop of (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} upon reoxygenation suggests a shift of metabolism to mitochondrial respiration when the glycolytic capacity in PTE was depleted. Our data clearly demonstrate that (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} elevation in PTE is not simply a postmortem event but an early change of anoxic injury. Loss of mitochondrial membrane potential accompanying elevated (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} implies a deleterious effect of sustained high (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} on mitochondria. Acidosis during ischemia has been recognized but the change in {dollar}pH\sb{lcub}i{rcub}{dollar} has not been reported in cultured PTE. Our results show that exposure to chemical anoxia causes the {dollar}pH\sb{lcub}\rm i{rcub}{dollar} to drop immediately, followed by spontaneous alkalinization. Immediately after this, {dollar}pH\sb{lcub}i{rcub}{dollar} continuously declined and remained low until rising moderately just before cell death. Parallel estimations of (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} shows increase of (Ca{dollar}\sp{lcub}2+{rcub}{dollar}) {dollar}\sb{lcub}i{rcub}{dollar} as {dollar}pH\sb{lcub}i{rcub}{dollar} declined. Prolongation of intracellular acidosis and protection were produced by blocking the {dollar}\rm Na\sp+/H\sp+{dollar} exchange activity with amiloride. Monensin, on the other hand, accelerated anoxic cytotoxicity. The terminal {dollar}pH\sb{lcub}\rm i{rcub}{dollar} rise may represent a phenomenon of cell permeability and irreversible cell injury. Taken together, our data suggested that the cytoprotective effects of acidosis may be to counteract Ca{dollar}\sp{lcub}2+{rcub}{dollar}-dependent mechanisms of anoxic injury.