Although ethanol abuse is the most common cause of pancreatitis, the mechanism of alcohol's effect on the pancreas is not well understood. Previously, we demonstrated that in vitro ethanol treatment of pancreatic acinar cells augmented the CCK-8-induced activation of NF-κB, a key signaling system involved in the inflammatory response of pancreatitis. In the present study, we determine the role for individual PKC isoforms in the sensitizing effect of ethanol on NF-κB activation. Dispersed rat pancreatic acini were treated with and without ethanol and then stimulated with CCK-8; 100 nM CCK-8 caused both NF-κB and PKC-δ, -ε, and -ζ activation, whereas 0.1 nM CCK-8 did not increase PKC-ε, PKC-ζ, or NF-κB activity. CCK-8 (0.1 nM) did activate PKC-δ. PKC-ε activator alone did not cause NF-κB activation; however, together with 0.1 nM CCK-8, it caused NF-κB activation. Ethanol activated PKC-ε without affecting other PKC isoforms or NF-κB activity. Of note, stimulation of acini with ethanol and 0.1 nM CCK-8 resulted in the activation of PKC-δ, PKC-ε, and NF-κB. The NF-κB activation to 0.1 nM CCK-8 in ethanol-pretreated acini was inhibited by both PKC-δ inhibitor and PKC-ε inhibitor. Taken together, these results demonstrate the different modes of activation of PKC isoforms and NF-κB in acini stimulated with ethanol, high-dose CCK-8, and low-dose CCK-8, and furthermore suggest that activation of both PKC-ε and -δ is required for NF-κB activation. These results suggest that ethanol enhances the CCK-8-induced NF-κB activation at least in part through its effects on PKC-ε.