Following our previous observations that anoxia induces a drop in extracellular Na+ in the brain slice and that removal of extracellular Na+ prevents the anoxia-induced morphological changes in dissociated hippocampal neurons, we hypothesized that intracellular Na+ increases during anoxia in isolated neurons. Using the fluorophore Sodium Green in freshly dissociated rat CA1 neurons, and SBFI in cultured cortical neurons, we found that 10 min of anoxia caused an increase in Nai+ in both types of cells, with a latency of about 2 min. In CA1 neurons, fluorescence increased by an average of 20.34% (n = 8). The mean baseline Nai+ level (determined using SBFI) was 25 +/- 2.5 mM, which increased to about an average of 52 +/- 3 mM after 3-4 min. These and our previous results strongly suggest that Na(+)-mediated events are involved in anoxia-induced nerve injury.