The aim of this study was to determine if protein kinase C- (PKC-) is involved in the repair of mitochondrial function and/or active Na⁺ transport following oxidant injury in renal proximal tubular cells (RPTC). Sublethal injury was produced in primary cultures of RPTC using tert-butylhydroperoxide (TBHP) and the recovery of functions was examined. PKC- was activated 3-5-fold after injury. Active PKC- translocated to the mitochondria. Basal oxygen consumption (QO₂), uncoupled QO₂, and ATP production decreased 58%, 60% and 41%, respectively, at 4 hr and recovered by day 4 after injury. At 4 hr, complex I-coupled respiration decreased 50% but complex II- and IV-coupled respirations were unchanged. Inhibition of PKC- translocation using a peptide selective inhibitor, PKC-V1-2, reduced decreases in basal and uncoupled QO₂s and increased complex I-linked respiration in TBHP-injured RPTC at 4 hr of recovery. Furthermore, PKC-V1-2 prevented decreases in ATP production in injured RPTC. Na⁺/K⁺-ATPase activity and ouabain-sensitive ⁸⁶Rb⁺ uptake were decreased by 60% and 53%, respectively, at 4 hr of recovery. Inhibition of PKC- activation prevented decline in Na⁺/K⁺-ATPase activity and reduced decreases in ouabain-sensitive ⁸⁶Rb⁺ uptake. We conclude that during early repair following oxidant injury in RPTC: 1) PKC- is activated and translocated to mitochondria, 2) PKC- activation decreases mitochondrial respiration, electron transport rate, and ATP production by reducing complex I-linked respiration, and 3) PKC- mediates decreases in active Na⁺ transport and Na⁺/K⁺-ATPase activity. These data show that PKC- activation following oxidant injury in RPTC is involved in the decreases in mitochondrial function and active Na⁺ transport and that inhibition of PKC- activation promotes the repair of these functions.