In the human astroglioma cell line CH235‐MG, interleukin‐1β (IL‐1β) induces transcriptional activation of the tumor necrosis factor‐alpha (TNF‐α) gene, resulting in expression of TNF‐α mRNA and biologically active TNF‐α protein. This study was undertaken to elucidate intracellular signaling pathways involved in IL‐1β induction of the TNF‐α gene. We demonstrated that the protein kinase C (PKC) activator 4β‐phorbol 12β‐myristate 13α‐acetate (PMA) in concert with Ca++ ionophore A23187 induced expression of TNF‐α mRNA and protein, whereas an inactive PMA analogue (αPMA) had no effect. Various cyclic nucleotide activators such as 8‐Bromo cAMP, cholera toxin, and forskolin had no effect on TNF‐α production. Two PKC inhibitors, H7 and staurosporine (SS), abrogated IL‐1β induced TNF‐α expression in a dose‐dependent fashion. Treatment of CH235‐MG cells with a high concentration of PMA (1 μM) for an extended period of time (48 h) caused a greater than 90% reduction in total PKC activity. Further strengthening a role for PKC in this cytokine response is the fact that IL‐1β was no longer able to induce TNF‐α expression in these PKC depleted cells. Last, IL‐1β treatment produced an increase of total PKC activity in CH235‐MG cells. Taken together, these data demonstrate that IL‐1β induces TNF‐α gene expression in CH235‐MG cells in a PKC‐dependent manner. © 1992 Wiley‐Liss, Inc. Copyright © 1992 Wiley‐Liss, Inc.