Protein kinase C (PKC) has been implicated in tumor necrosis factor-α (TNF) signaling. Structurally and functionally distinct PKC activators and selective inhibitors of PKC were used to investigate the involvement of PKC isozymes in influencing TNF sensitivity in MCF-7 cells. Activators of PKC, such as phorbol-12,13-dibutyrate (PDBu) (1.0 μM), indolactam V (10 μM), and bryostatin 1 (1.0 μM) decreased the sensitivity of MCF-7 cells to TNF by 5- , 10-, and 1.7-fold, respectively. The PKC-specific inhibitor bisindolylmalelmide II (BIM) (≤1 μM) antagonized the effect of PDBu in protecting MCF-7 cells against TNF cytotoxicity. High concentrations of BIM (≤10 μM) also significantly enhanced the sensitivity of MCF-7 cells to TNF. In contrast, Go 6976, a specific inhibitor of cPKCs, did not potentiate TNF sensitivity and failed to reverse the effect of PDBu. In addition, BIM but not Go 6976 blocked PDBu-mediated down-regulation of TNF receptors. There was no correlation between down-regulation of PKCα, -δ, and -ε, and protection against TNF cytotoxicity by PKC activators. A 6-hr exposure to 1.0 PDBu, 10 μM indolactam V, and 1.0 μM bryostatin 1 caused a 1.8-, 3.5- and 1.2-fold induction, respectively, of nPKCη in MCF-7 cells. Similar exposure to BIM but not Go 6976 led to a significant down-regulation of nPKCη. This novel regulation of PKCη implicates this isozyme in PDBu-mediated protection of MCF-7 cells against TNF cytotoxicity.