Cardiac ventricular myocytes from several species, including the guinea pig, possess a cAMP-dependent protein kinase A (PKA)-activated Cl− channel. In the present study, the properties of a protein kinase C (PKC)-activated Cl− current were studied in isolated guinea pig ventricular myocytes using the whole-cell arrangement of the patch-clamp technique. Intracellular dialysis of ventricular cells with PKC resulted in the activation of a large background current that displayed time- independent kinetics. In the presence of 146 mmol/L external Cl− and 71 mmol/L internal Cl−, the reversal potential (Erev) of the background current (−17±1 mV) was close to that of the Cl− equilibrium potential (−18 mV), and the current versus voltage relation for the current was outward rectifying in shape. When [Cl−]i or [Cl−]o was reduced by substitution of Cl− with aspartic acid, Erev, for the background current shifted in a manner expected for a Cl− selective channel. Based on Erev, measurements, the permeability sequence for this PKC-activated Cl− channel was determined to be SCN− >I− >Br− ≃ Cl−. The PKC-activated Cl− current was not inhibited by the Cl− channel blocker 4,4′ -dinitrostilbene-2,2′-disulfonic acid (100 μmol/L) but could be blocked by anthracene-9-carboxylic acid (1 mmol/L). Activation of the current was abolished in the presence of the PKC inhibitor staurosporine (2.5 μmol/L). Under conditions designed to cause a maximal activation of the Cl− channels by PKC, the addition of forskolin (1 μmol/L) to stimulate PKA caused only a slight further increase in the amplitude of the Cl− current. Thus, PKC activates a Cl− channel in guinea pig ventricular cells with properties similar but not identical to the PKA-activated channel.