A model of in vitro traumatic injury with dissociated rat hippocampal neurons was studied to explore the mechanism of cell death. The neurotoxicity induced by traumatic injury to the cell culture can be transferred to a naive uninjured culture by media exchange. This toxicity is attenuated by dimethylsulfoxide or superoxide dismutase, suggesting that this toxicity is mediated by a free radical generation. Ionotropic glutamate receptor antagonists had no effect. This toxicity was effectively blocked by the pretreatment of the naive uninjured recipient cultures with cycloheximide or with actinomycin D. The DNA fragmentation could be illustrated with in situ nick translation in the cells which seem to have lost their cytoplasm. The nuclear morphology of neurons labeled by a neurofilament-specific antibody, SMI-31, demonstrated chromatin condensation and nucleosome formation. Traumatic injury induces release of an unknown toxin into the extracellular space. These observations suggest that a traumatized neuronal culture can propagate cell death of naive uninjured cells by releasing a neurotoxin that causes apoptosis.