Tumour necrosis factor (TNF) is a key regulator of inflammation and immunity. The cellular effects exerted by TNF depend, apart from NF-κB-directed gene transcription, largely on its ability to activate phospholipase A₂(PLA₂), yielding the release of arachidonic acid (AA) and its metabolites. AA metabolites, especially the leukotrienes, act as second messengers in TNF receptor signalling, as different inhibitors of AA metabolism impair a variety of TNF-induced biochemical events. The role, however, of AA and its metabolites in TNF-induced NF-κB activation is still obscure. Here we report that 4-bromophenacyl bromide (4-BPB; an inhibitor of PLA₂), nordihydroguaretic acid (NDGA; a 5-lipoxygenase inhibitor), as well as MK-886 [an inhibitor of 5-lipoxygenase-activating protein (FLAP)] interfere with TNF-induced NF-κB-mediated transactivation. However, only 4-BPB inhibited the DNA-binding activity of NF-κB, whereas NDGA and MK-886 did not. Thus, different inhibitors interfere at different points in TNF-induced signalling leading to NF-κB-dependent transcription. Artificial induction of AA metabolism induced neither DNA-binding activity of NF-κB nor NF-κB-dependent transactivation. It was concluded that although TNF-induced signalling to NF-κB-dependent transcription is sensitive to inhibitors of AA metabolism at multiple points during this signalling, AA release is essential but not sufficient for NF-κB-activation.