Physiological increases in liver cell volume lead to an adaptive response that includes opening of membrane Cl− channels, which is critical for volume recovery. The purpose of these studies was to assess the potential role for protein kinase C (PKC) as a signal involved in cell volume homeostasis. Studies were performed in HTC rat hepatoma and Mz-ChA-1 human cholangiocarcinoma cells, which were used as model hepatocytes and cholangiocytes, respectively. In each cell type, cell volume increases were followed by: 1) translocation of PKCα from cytosolic to particulate (membrane) fractions; 2) a 10- to 40-fold increase in whole-cell membrane Cl− current density; and 3) partial recovery of cell volume. In HTC cells, the volume-dependent Cl− current response (−46 ± 5 pA/pF) was inhibited by down-regulation of PKC (100 nmol/L phorbol 12-myristate 13-acetate for 18 hours [PMA]; −1.97 ± 1.5 pA/pF), chelation of cytosolic Ca2+ (2 mmol/L EGTA; −5.3 ± 4.0 pA/pF), depletion of cytosolic adenosine triphosphate (ATP) (3 U/mL apyrase; −12.58 ± 1.45 pA/pF), and by the putative PKC inhibitor, chelerythrine (25 μmol/L; −7 ± 3 pA/pF). In addition, PKC inhibition by chelerythrine and calphostin C (500 nmol/L) prevented cell volume recovery from swelling. Similar results were obtained in Mz-ChA-1 biliary cells. These findings indicate that swelling-induced activation of PKC represents an important signal coupling cell volume to membrane Cl− permeability in both hepatic and biliary cell models.