The transcription factor activator protein-1 (AP-1) is activated in response to physiological activity in neuronal circuits and in response to neuronal injury associated with various acute and chronic neurodegenerative conditions. The membrane lipid peroxidation product 4-hydroxy-2,3-nonenal (HNE) is increasingly implicated in the disruption of neuronal calcium homeostasis that occurs in various paradigms of neuronal excitotoxicity and apoptosis. The possible mechanistic links between lipid peroxidation and alterations in gene transcription during neuronal apoptosis have not previously been examined. We now report that exposure of cultured rat cortical neurons to an apoptotic concentration of HNE results in a large increase in AP-1 DNA-binding activity. The protein synthesis inhibitor cycloheximide blocked the induction of AP-1, consistent with a requirement for induction of expression of AP-1 family members. The broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde blocked HNE-induced increases in AP-1 DNA-binding activity, demonstrating a requirement for caspase activation in the activation of AP-1. HNE induced phosphorylation of c-Jun N-terminal kinase (JNK), which was prevented by caspase inhibitors, indicating that HNE was acting at or upstream of JNK phosphorylation. The intracellular calcium chelator BAPTA-acetoxymethyl ester completely prevented stimulation of AP-1 DNA-binding by HNE, indicating a requirement for calcium. Moreover, agents that suppress mitochondrial calcium uptake (ruthenium red) and membrane permeability transition (cyclosporin A) attenuated AP-1 activation by HNE, suggesting a contribution of mitochondrial alterations to AP-1 activation. Collectively, our data suggest a scenario in which HNE disrupts neuronal calcium homeostasis and perturbs mitochondrial function, resulting in caspase activation. Activated caspases, in turn, induce activation of JNK, resulting in stimulation of AP-1 DNA-binding protein production. This transcriptional pathway induced by HNE may modulate the cell death process.