Volume-sensitive outwardly rectifying (VSOR) Cl- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl- channel in cardiomyocytes, the properties of VSOR Cl- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3(-/-)) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3(+/+)) and heterozygous (Clcn3(+/-)) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl- currents. We thus conclude that functional expression of VSOR Cl- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.