C-Jun N-terminal kinase (JNK), a member of the MAPK family of protein kinases is a stress-response kinase that is activated by pro-inflammatory cytokines and growth factors coupled to membrane receptors or through non-receptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase Pyk2 and the small GTP binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase-c (PKC) and TGF- activated kinase 1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-/-selective inhibitor Go9367 or adenovirus expressing dominant negative PKC- blocked the phosphorylation of Pyk2 and JNK. Reoxygenation activated the MAP-KKK, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1 and JNK but not ASK-1 or PAK-1, that was detected within the first 10 minutes of reoxygenation. These results identify 2 new components, PKC and TAK-1 that have not been previously described in this signaling pathway.