The supporting role of glial cells in maintaining neurons and in ion homeostasis has been studied in situ by perfusing the gliotoxin fluorocitrate (FC) through a microdialysis fiber in the CA1 area of urethane-anesthetized rats. Extracellular direct current potential, extracellular potassium concentration ([K+]o) and amino acid levels, extracellular pH (pHo), and evoked field activity were studied. Histology verified the swelling of glial cells after 4 hr of FC treatment. Massive neuron damage was evident after 8 hr. FC dialysis caused the rapid decrease of glutamine, pHo became progressively more acid, and [K+]o moderately elevated. Orthodromic transmission was variably blocked within 30 min to 4 hr. After 4 hr, spreading depression (SD) waves that originated from the neocortex invaded hippocampal CA1, [K+]o increased to higher levels, pHo became very acid, and there were steep increases in taurine, glutamate, and GABA levels. Simultaneously, the antidromic population spike (a-PS) became depressed and eventually disappeared. When a shorter dialysis probe that spared cortex was used to sample CA1, no SD was seen, a-PS was not abolished, and ion homeostasis was altered less markedly. Repeated SD provoked in hippocampus in the absence of FC caused only mild depression of a-PS. Dialysis of high-K+ solution in healthy neocortex or hippocampus caused only slight elevation of [K+]o at distances of 200–400 microns from the dialysis membrane. After treatment with FC, similar high-K+ dialysis raised [K+]o much more. We conclude the following: (1) recurrent SD waves injure neurons if and only if glial function has failed; (2) neurons can regulate [K+]o, albeit imperfectly; (3) glia is required for the normal fine tuning of [K+]o and particularly for the recovery of pathologically elevated [K+]o; and (4) glia are required for the regulation of pHo. The similarities between glial poisoning by FC and the reported changes in the penumbra of ischemic infarcts suggest that the extension of neuron loss into the penumbral region might depend on failure of glial protection.