C-Jun N-terminal kinase 1 and 2 (JNK1/2) have been shown to be transiently activated and involved in neurotoxicity. We searched for possible upstream molecules, which are responsible for the regulation of hydrogen peroxide-(H2O2) induced JNK1/2 activation and JNK1/2-mediated apoptotic-like cell death in cultured rat cortical neurons. The results showed that JNK1/2 activation (monitored by anti-diphosphorylated JNK1/2 antibody) was largely prevented by elimination of extracellular Ca2+ or blockage of NMDA-receptors (NMDA-R), and was weakly but significantly decreased by blockage of L-type voltage-gated calcium channel (L-VGCC); furthermore, JNK1/2 activation was largely prevented by inhibition of Ca2+/calmodulin-dependent protein kinase-II (CaMKII) and protein-tyrosine kinases (PTK). We also found that H2O2-induced apoptotic-like cell death was partially prevented by elimination of extracellular Ca2+, or by inhibition of NMDA-R, L-VGCC, PTK and CaMKII, respectively. The above results suggest that in H2O2-induced neurotoxicity, JNK1/2 activation is mainly mediated by NMDA-R and L-VGCC. Consequently, PTK and CaMKII are critical intermediaries in JNK1/2 activation and are mainly responsible for JNK1/2-mediated apoptotic-like cell death.