Bacterial endotoxin lipopolysaccharide (LPS) treatment of neuron-rich cells and glia-rich cells exhibited significant cell damage 12 hr after incubation, although no severe or significant cell damage induced by LPS appeared in neuron-glia co-cultured cells. Moreover, severe and significant time-dependent cell damage was induced by a larger dose treatment (10 mM) of glutamate (Glu), and this damage was seen in neuron-rich cells, neuron-glia co-cultured cells, and glia-rich cells. Examining extracellular tumor necrosis factor alpha (TNFalpha) induced by either LPS or Glu treatment, the levels of extracellular TNFalpha induced by LPS were significantly higher than those induced by Glu. These significant increases of TNFalpha were measured within 2 hr after LPS treatment in neuron-glia co-cultured cells and glia-rich cells, although no significant changes were detected in the neuron-rich cells. With Glu treatment, a significant increase in TNFalpha levels was detected after 6 hr of Glu treatment only in glia-rich cells. Our results indicate that cerebral TNFalpha is mainly produced in glia cells and that its production is dependently regulated by each stimulant. In addition, the production of TNFalpha is not directly related to the trigger of cell injury.