Previously, we have shown that oxidant exposure in renal proximal tubular cells (RPTC) induces mitochondrial dysfunction mediated by PKC-. This study examined role of ERK1/2 in mitochondrial dysfunction induced by oxidant injury and whether PKC- mediates its effects on mitochondrial function through the Raf-MEK1/2-ERK1/2 pathway. Sublethal injury produced by tertbutylhydroperoxide (TBHP) resulted in 3-5-fold increase in phosphorylation of ERK1/2 and p38 but not JNK. This was followed by decreases in basal and uncoupled respirations (41%), state 3 respiration and ATP production coupled to complex I (46%), and complex I activity (42%). Oxidant exposure decreased aconitase activity 30% but not pyruvate, -ketoglutarate, and malate dehydrogenase activities. Inhibition of ERK1/2 restored basal and state 3 respirations, _m, ATP production, and complex I activity but not aconitase activity. In contrast, activation of ERK1/2 by expression of constitutively active MEK1 suppressed basal, uncoupled, and state 3 respirations in non-injured RPTC to the levels observed in TBHP-injured RPTC. MEK1/2 inhibition did not change Akt or p38 phosphorylation demonstrating that the protective effects of MEK1/2 inhibitors were not due to activation of Akt or inhibition of p38 pathways. Inhibition of PKC- did not block TBHP-induced ERK1/2 phosphorylation in whole RPTC or in mitochondria. We conclude that: 1) oxidant-induced activation of ERK1/2 but not p38 or JNK reduces mitochondrial respiration and ATP production by decreasing complex I activity and substrate oxidation through complex I, 2) citric acid cycle dehydrogenases are not under control of the ERK1/2 pathway in oxidant-injured RPTC, 3) the protective effects of ERK1/2 inhibition are not due to activation of Akt, and 4) ERK1/2 and PKC- mediate oxidant-induced mitochondrial dysfunction through independent pathways.