We examined cytokine‐mediated neuronal death in neuron‐astrocyte cultures from second trimester human fetal cerebrum. In these cultures, high‐output inducible nitric oxide synthase (NOS) and tumor necrosis factor‐α (TNFα) are expressed in astrocytes after exposure to IL‐1β/IFNγ. Neuronal cell death was evident at ≥48 h following cytokine stimulation. Neutralizing anti‐TNFα antiserum inhibited (≈48%) neurotoxicity in IL‐1β/IFNγ‐treated cultures, demonstrating a role for endogenously produced TNFα. Interestingly, the degree of neuroprotection conferred by superoxide dismutase or N‐methyl D‐aspartate (NMDA) receptor antagonists in these cultures was smaller and variable. Similarly, the effect of the NOS inhibitor, N^G‐monomethyl L‐arginine (NMMA) on IL‐1β/IFNγ‐induced neuronal death was variable, showing no statistically significant effect when results from more than 30 independent cultures were averaged. Neurons die by apoptosis in cytokine‐treated human fetal CNS cultures as shown by the characteristic nuclear morphology as well as positive labeling for TUNEL. Our results demonstrate a potent neurotoxicity mediated by the cytokine combination IL‐1β/IFNγ in primary human neuron‐astrocyte cultures and a crucial role for endogenous TNFα in mediating neurotoxicity in this system. These results firmly establish the neurotoxic potential of the inflammatory cytokines IL‐1β and TNFα in the human CNS. GLIA 28:114–127, 1999. © 1999 Wiley‐Liss, Inc.