[Truncated abstract] Reactive oxygen species (ROS) have been implicated to play a major role in many pathological conditions including heart attack and stroke. Their ability to modulate the extracellular signal-regulated protein kinase (ERK) and c-Jun Nterminal kinase (JNK) signalling pathways, thereby influencing cellular response has been well-documented. Recent studies implicate a central role for mitochondria in ERK and JNK activation by ROS although the mechanisms remained unresolved. Using Jurkat T-lymphocyte as a cell model, this study demonstrated increased mitochondrial ROS production as a result of decreased mitochondrial complex activities mediated by hydrogen peroxide treatment. This is the first study to show that mitochondria are not essential for activating ERKs, however damaged mitochondria producing ROS can be expected to cause sustained ERK activation . . . This study revealed that JNK and its upstream kinases MKK4, MKK7 and ASK1 are associated with the mitochondria. Furthermore, findings from this study imply that JNK resides in the mitochondrial matrix. This study is the first to demonstrate that mitochondrial JNK can be activated in a cell-free environment by signals originating from the mitochondria. Experimental work using isolated mitochondria demonstrated that mitochondrial JNK can be activated by ROS generated from the mitochondria themselves. Flavin-containing proteins appear to be the main sources of mitochondrial-ROS which signal through redoxsensitive proteins to activate mitochondrial JNK.