The present study was undertaken to explore the role of interleukin‐12 (IL‐12) p40 in the expression of TNF‐α in microglia. Interestingly, we have found that IL‐12 p70, p40₂ (the p40 homodimer) and p40 (the p40 monomer) dose‐dependently induced the production of TNF‐α and the expression of TNF‐α mRNA in BV‐2 microglial cells. In addition to BV‐2 microglial cells, p70, p40₂ and p40 also induced the production of TNF‐α in mouse primary microglia and peritoneal macrophages. As the activation of both NF‐κB and CCAAT/enhancer binding protein β (C/EBPβ) is important for the expression of TNF‐α in microglial cells, we investigated the effect of p40 on the activation of NF‐κB as well as C/EBPβ. Activation of NF‐κB as well as C/EBPβ by p40 and inhibition of p40‐induced expression of TNF‐α by Δp65, a dominant‐negative mutant of p65, and ΔC/EBPβ, a dominant‐negative mutant of C/EBPβ, suggests that p40 induces the expression of TNF‐α through the activation of NF‐κB and C/EBPβ. In addition, we show that p40 induced the activation of both extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK). Interestingly, PD98059, an inhibitor of ERK, inhibited p40‐induced expression of TNF‐α through the inhibition of C/EBPβ, but not that of NF‐κB, whereas SB203580, an inhibitor of p38 MAPK, inhibited p40‐induced expression of TNF‐α through the inhibition of both NF‐κB and C/EBPβ. This study delineates a novel biological function of p40 in inducing TNF‐α in microglia and macrophages.