The consequences of activation of tumour necrosis factor receptor 1 (TNFR1) during neuronal injury remain controversial. The apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, can mediate cell death downstream of TNFR1. Presently, we examined the formation of the TNFR1 signalling cascade and response of ASK1 during seizure-induced neuronal death. Brief (40 min) seizures were induced in rats by intra-amygdala microinjection of kainic acid, which elicited unilateral hippocampal CA3 neuronal death. Seizures caused a rapid decline in the expression of the silencer of death domains protein within injured CA3. Co-immunoprecipitation analysis revealed a commensurate assembly of a TNFR1 scaffold complex containing TNFR-associated death domain protein, receptor interacting protein and TNFR-activating factor 2. In addition, recruitment of TNFR-activating factor 2 was likely promoted by Bcl10-mediated sequestering of cellular inhibitor of apoptosis protein 2. Apoptosis signal-regulating kinase 1 was sequestered in a complex that contained the molecular chaperone 14-3-3beta and protein phosphatase 5. Seizures triggered its dissociation, and the phosphorylation of the ASK1 substrates, mitogen-activated protein kinase kinase 3/6 and 4. Subsequently, protein phosphatase 5 translocated into the nuclei of degenerating CA3 neurons, while ASK1 colocalized with the adaptor proteins Daxx and TNFR-activating factor 2 at the outer membrane of injured CA3 neurons. Neutralizing antibodies to TNFalpha reduced the numbers of DNA damaged cells within the injured hippocampus. These data suggest ASK1 may be involved in the mechanism of seizure-induced neuronal death downstream of a TNFR1 death-signalling complex.