Bacterial infection has been implicated in the selective brain white matter injury commonly found in cerebral palsy, a major birth disorder. We have examined the direct toxicity of the bacterial endotoxin lipopolysaccharide (LPS) upon glial cells in perfused, isolated optic nerve of humanely killed 8-12 day post-natal rat (a white matter tract). Nerves were dissected and glial cells loaded in situ with the Ca²⁺ -sensitive dye FURA-FF. Nerves were continually perfused with ACSF and imaged using a standard monochrometer based set-up. Exposure to LPS (1 μg/ml) produced cell death in 59.4 ±10.6% (mean ±SEM) of glia (n=63 cells in 3 nerves) over a 180 min time-course, determined by step-wise loss of FURA-FF fluorescence. 1 out of 53 glial cells in 3 nerves died during 180 min of control (no LPS) perfusion (P<0.001 by ANOVA). A delayed [Ca²⁺]_i rise preceded the cell death induced by LPS and both death and the Ca2+-rise were prevented by removing Ca²⁺_o| (+ 50 μM EGTA), or blocking ionotropic glutamate receptors with either CNQX (20 μM) or MK-801 (50 μM). In a separate set of experiments, ethidium bromide was used to stain dead cell nuclei in optic nerves after a 180 min incubation under various conditions. The number of dead cells was increased to 139 ±5.4% (n=9 nerves) in nerves incubated in LPS compared to those in ACSF alone (P<0.001). Tumor necrosis factor-α (TNF-α; 1000 U/ml) or interleukin-1β (1000 U/ml) mimicked this cytotoxic effect of LPS. An increase in the number of dead cells to 141 ±4.9% (n=8) was observed following incubation in TNF-α (P<0.005), and to 136 ±5.4% (n=9 nerves) following interleukin-1 incubation (P<0.005). Either removing the biological activity of TNF-α with a blocking antibody, or co-application of a recombinant antagonist of interleukin-1, prevented the cytotoxic action of LPS. A mix of reactive oxygen species scavengers (500 μM Tempo + 250 U/ml catalase) also blocked the cell death induced by LPS. LPS is therefore directly and acutely toxic to developing white matter glial cells via a mechanisms involving cytokine and ROS production, glutamate receptor activation and Ca²⁺-influx.