We report the characterization of the first successful treatment of neuronal ischemic injury in vivo by cell-permeant Ca2+ chelators. The chelators attenuated glutamate-induced intracellular Ca2+ increases and neurotoxicity in neuronal explant cultures. When infused intravenously in rats, permeant fluorescent BAPTA analogs accumulated in neurons in several brain regions. BAPTA-AM, infused in vivo, reduced Ca(2+)-dependent spike frequency adaptation and post-spike train hyperpolarizations in CA1 neurons taken from treated animals. This effect was reproduced by direct injections of BAPTA into untreated neurons. The effects of three different chelators (BAPTA, 5,5'-difluoro BAPTA, and 4,4'-difluoro BAPTA) on Ca(2+)-dependent membrane excitability varied with their Ca2+ affinity. When the chelators' permeant forms were used to treat rats prior to the induction of focal cortical ischemia, they were highly neuroprotective, as gauged by significant reductions in cortical infarction volumes and neuronal sparing. The chelators' protective effects in vivo also reflected their affinity for Ca2+. This report provides the most direct evidence to date that intracellular Ca2+ excess triggers early neurodegeneration in vivo and contributes a novel therapeutic approach to neuronal ischemia of potential clinical utility.