The effects of metabolic acidosis were examined in isolated rat hepatocytes under substrate-free oxygenated or anoxic conditions. Lowering extracellular pH to 6.6 under aerobic conditions had no deleterious effects on the cells as determined by trypan blue exclusion, lactate dehydrogenase (LDH) release, cellular K+ and Ca2+ content, and ability to increase ATP levels after nutrients and adenosine were added to media. Cytosolic pH was measured in aerobic cells at varying extracellular pH using 6-carboxyfluorescein. By using values for cytosolic pH obtained in this manner together with 5,5-dimethyl[2-14C]oxazolidine-2,4-dione (DMO) distribution data, a method was derived for determining intramitochondrial pH. The pH gradient across the mitochondrial membrane was found not to change with a decrease in extracellular pH from 7.4 to 6.9. At pH 6.9 hepatocytes were protected against anoxic injury as compared with cells incubated at pH 7.5 or 6.6. This protection was manifested by a decrease in vital dye uptake and LDH release, maintenance of higher cellular K+ content, less stimulation of respiration with succinate, improved recovery of ATP levels after return to an oxygenated nutrient environment, and maintenance of normal cellular Ca2+ content after reoxygenation. Recovery of cellular ATP content was independent of ATP levels, total adenine nucleotide pool, and energy charge ratio at the end of the anoxic period. Measurement of cytoplasmic pH in anaerobic cells by [14C]DMO distribution showed progressive cellular acidification with lowering of extracellular pH. The protective effects observed at pH 6.9 are not unique to hepatocytes since isolated renal cortical tubules exposed to anoxia have improved ATP levels on reoxygenation at this pH when compared with tubules incubated at pH 7.5.