This study measures cellular lactate dehydrogenase (LDH) release during metabolic inhibition as a monitor of sarcolemmal integrity as affected by variation of external pH (pH_e) and Ca²⁺ concentration ([Ca²⁺]_e). The sigmoidal relationship between pH_e and LDH release and pH_e and net Ca²⁺ uptake was essentially identical with the 50% maximal value occurring at pH 7.0 for both. This suggests that a process(es) sensitive to both pH_e and [Ca²⁺]_e plays a role in cell lysis during the course of metabolic inhibition. Variation of pH_e during metabolic inhibition did not alter the decline in cellular ATP, nor did it affect changes in sarcolemmal phospholipid topology. Intracellular pH followed changes of pH_e with a few minutes lag. Cell lysis increased in a graded manner as pH_e and [Ca²⁺]_e were increased, but pH_e was the sole determinant of lysis, i.e., [Ca²⁺]_e level had no effect, at the lowest (6.2) and the highest (8.0) pH_e levels. pH_e variation did not affect the release of radiolabeled arachidonic acid, nor did inhibitors of phospholipase A₂ (PLA₂) affect cell lysis at varying pH_e. Therefore, cellular PLA₂ activation could not be implicated for a role in cell lysis in the present model of metabolic inhibition. Alternatively, we propose that Ca²⁺ binding to the cytoplasmic leaflet, in combination with membrane alterations secondary to the metabolic insult, combine to destabilize the sarcolemma (20). This Ca²⁺ binding to the negatively charged phosphatidylserine results in the expression of the bilayer destabilizing effect of phosphatidylethanolamine. This Ca²⁺ binding is greatly diminished by lowered pH, resulting in an attenuation of cell lysis.