Lowering extracellular pH to less than 7.0 strongly protects isolated proximal tubules against ATP depletion and Ca2+-induced injury, but there is little information about alterations of intracellular pH (pH(i)) in renal tubules during either injury or its modification by decreasing medium pH or other potent protective factors such as glycine. pH(i) was assessed with 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein during proximal tubule injury produced by simple ATP depletion with the electron transport inhibitor antimycin or by large increases of cytosolic free Ca2+ induced by treatment with the calcium ionophore iono mycin, alone and in combination with antimycin. Freshly isolated rabbit proximal tubules studied under superfusion conditions in the presence of probenecid were suitable for monitoring pH(i) during relatively prolonged and severe injury states. Probenecid, used to promote the retention of intracellular fluorophores, only minimally modified the injury response by transiently delaying lactate dehydrogenase release during antimycin treatment. The tubules did not exhibit spontaneous decreases of pH, during simple ATP depletion, but pH(i) fully equilibrated with cytoprotective decreases of medium pH. Irrespective of the presence of antimycin, ionomycin induced intracellular alkalinization in Ca2+-replete medium, which may have further enhanced the severity of injury. When medium Ca2+ was buffered to 100 nM, ionomycin induced intracellular acidification, which likely resulted from a combination of Ca2+/H+ exchange activity of the ionophore and H+ uptake during Ca2+-ATPase-mediated extrusion of Ca2+ released by ionomycin from intracellular pools. Alterations of pH(i) did not contribute to glycine cytoprotection because glycine did not affect the behavior of pH(i) during treatment with antimycin, ionomycin, or both agents in combination. The lack of effect of glycine on pH(i) makes it likely that complementary cytoprotective actions of glycine and reduced pH will be relevant to understanding both in vitro models of injury and in vivo ischemic states.