Previously, we have shown that physiological functions of renal proximal tubular cells (RPTC) do not recover following S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced injury. This study investigated the role of protein kinase C- (PKC-) in the lack of repair of mitochondrial function in DCVC-injured RPTC. Following DCVC exposure, basal oxygen consumption (QO₂), uncoupled QO₂, oligomycin-sensitive QO₂, F₁F₀-ATPase activity and ATP production decreased, respectively, to 59%, 27%, 27%, 57% and 68% of controls. None of these functions recovered. Mitochondrial transmembrane potential decreased 53% after DCVC injury but recovered on day 4. PKC- was activated 4.3- and 2.5-fold on days 2 and 4, respectively, of the recovery period. Inhibition of PKC- activation (10 nM Go6976) did not block DCVC-induced decreases in mitochondrial functions but promoted the recovery of uncoupled QO₂, oligomycin-sensitive QO₂, F₁F₀-ATPase activity, and ATP production. Protein levels of the catalytic subunit of F₁F₀-ATPase were not changed by DCVC or during the recovery period. Amino acid sequence analysis revealed that , , and subunits of F₁F₀- ATPase have PKC consensus motifs. Recombinant PKC- phosphorylated the subunit and decreased F₁F₀-ATPase activity in vitro. Serine but not threonine phosphorylation of the subunit was increased during late recovery following DCVC injury and inhibition of PKC- activation decreased this phosphorylation. We conclude that during RPTC recovery following DCVC injury: 1) PKC- activation decreases F0F1-ATPase activity, oxidative phosphorylation, and ATP production, 2) PKC- phosphorylates the subunit of F₁F₀-ATPase on serine residue, and 3) PKC- does not mediate depolarization of RPTC mitochondria. This is the first report showing that PKC- phosphorylates the catalytic subunit of F₁F₀-ATPase and that PKC- plays an important role in regulating repair of mitochondrial function.