A brief global ischemic insult to the brain leads to a selective degeneration of the pyramidal neurons in the hippocampal CA1 region while the neurons in the neighbouring CA3 region are spared. The reason for this difference is not known. The selective vulnerability of CA1 neurons to ischemia can be reproduced in vitro in murine organotypic slice cultures, if the ion concentrations in the medium during the anoxic/aglycemic insult are similar to that in the brain extracellular fluid during ischemia in vivo. As acidosis develops during ischemia, we studied the importance of extracellular pH for selective vulnerability. We found that cell death in the CA1 and CA3 regions was equally prevented by removal of calcium from the medium or following blockade of the N-methyl-D-aspartate (NMDA) receptor by D-2 amino-5-phosphonopentanoic-acid (D-APV). On the other hand, damage to the CA3 neurons markedly decreased with decreasing pH following in vitro ischemia, while the degeneration of CA1 neurons was less pH dependent. Patch-clamp recordings from pyramidal neurons in the CA1 and CA3 regions, respectively, revealed a pronounced inhibition of NMDA-receptor mediated excitatory postsynaptic currents (EPSCs) at pH 6.5 that was equally pronounced in the two regions. However, when changing pH from 6.5 to 7.4 the recovery of the EPSCs was significantly slower in the CA3 region. We conclude that acidosis selectively protects CA3 pyramidal neurons during in vitro ischemia, and differentially affects the kinetics of NMDA receptor activation, which may explain the difference in vulnerability between CA1 and CA3 pyramidal neurons to an ischemic insult.