In this study, we examined whether necrosis induced by H₂O₂ is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H₂O₂ treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus H₂O₂ treatment induces a "lag phase" where signaling changes occur including protein kinase C (PKC) activation, Akt (PKB) down regulation, activation of c-jun N-terminal kinase (JNK) and down regulation of AMP activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H₂O₂ (~ 80%). PKC inhibitor treatment decreased PKC activity, but surprisingly also upregulated Akt and AMPK, suggesting various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H₂O₂-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H₂O₂ even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H₂O₂. Furthermore, AMPK activators protected against H₂O₂-induced necrosis suggesting that much of the protective effect of PKC inhibition was mediated through upregulation of AMPK. Work with PKC inhibitors suggest that atypical PKC downregulates AMPK in response to H₂O₂. Knocking down PKC- using antisense also slightly protected (~22%) against H₂O₂. Taken together our data demonstrate that modulation of signaling pathways involving PKC and AMPK can alter H₂O₂-induced necrosis, suggesting a signaling "program" is important in mediating H₂O₂-induced necrosis in primary hepatocytes.