The ratiometric indicators 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and Fura-2 were employed to examine, respectively, intracellular pH (pH i) and calcium ([Ca 2+] i) changes evoked by anoxia in cultured postnatal rat hippocampal neurons at 37°C. Under both HCO − 3/CO 2- and HEPES-buffered conditions, 3-, 5- or 10-min anoxia induced a triphasic change in pH i consisting of an initial fall in pH i, a subsequent rise in pH i in the continued absence of O 2 and, finally, a further rise in pH i upon the return to normoxia, which recovered towards preanoxic steady-state pH i values if the duration of the anoxic insult was ≤5 min. In parallel experiments performed on sister cultures, anoxia of 3, 5 or 10 min duration evoked rises in [Ca 2+] i which, in all cases, commenced after the start of the fall in pH i, reached a peak at or just following the return to normoxia and then declined towards preanoxic resting levels. Removal of external Ca 2+ markedly attenuated increases in [Ca 2+] i, but failed to affect the pH i changes evoked by 5 min anoxia. The latency from the start of anoxia to the start of the increase in pH i observed during anoxia was increased by perfusion with media containing either 2 mM Na +, 20 mM glucose or 1 μM tetrodotoxin. Because each of these manoeuvres is known to delay the onset and/or attenuate the magnitude of anoxic depolarization, the results suggest that the rise in pH i observed during anoxia may be consequent upon membrane depolarization. This possibility was also suggested by the findings that Zn 2+ and Cd 2+, known blockers of voltage-dependent proton conductances, reduced the magnitude of the rise in pH i observed during anoxia. Under HCO − 3/CO 2-free conditions, reduction of external Na + by substitution with N-methyl- d-glucamine (but not Li +) attenuated the magnitude of the postanoxic alkalinization, suggesting that increased Na +/H + exchange activity contributes to the postanoxic rise in pH i. In support, rates of pH i recovery from internal acid loads imposed following anoxia were increased compared to control values established prior to anoxia in the same neurons. In contrast, rates of pH i recovery from acid loads imposed during anoxia were reduced, suggesting the possibility that Na +/H + exchange is inhibited during anoxia. We conclude that the steady-state pH i response of cultured rat hippocampal neurons to transient anoxia is independent of changes in [Ca 2+] i and is characterized by three phases which are determined, at least in part, by alterations in Na +/H + exchange activity and, possibly, by a proton conductance which is activated during membrane depolarization.