The ‘classical’ NF‐κB activation pathway proceeds via IκB kinase (IKK)‐β/γ‐mediated phosphorylation, induced ubiquitination and the degradation of small IκBs. An alternative, NF‐κB‐inducing kinase and IKK‐α‐dependent pathway, which stimulates the processing of NF‐κB2/p100, has recently been suggested. However, no physiological stimulus has been shown to trigger the activation of this pathway. Here we demonstrate that persistent stimulation with lymphotoxin β (LT‐β) receptor agonists or lipopolysaccharide (LPS), but not with interleukin‐1β, tumour necrosis factor‐α or 12‐O‐tetradecanoylphorbol‐13‐acetate, induces the generation of p52 DNA‐binding complexes by activating the processing of the p100 precursor. Induction of p52 DNA‐binding activity is delayed in comparison with p50/p65 complexes and depends on de novo protein synthesis. p100 is constitutively and inducibly polyubiquitinated, and both ubiquitination and p52 generation are coupled to continuing p100 translation. Thus, both LT‐β receptor agonists and LPS induce NF‐κB/p100 processing to p52 at the level of the ribosome.