Background: Protein kinase C (PKC) is a family of isoenzymes that regulate a variety of functions in the central nervous system including neurotransmitter release, ion channel activity, and cell differentiation. Growing evidence suggests that specific isoforms of PKC influence a variety of behavioral, biochemical, and physiological effects of ethanol in mammals. The purpose of this study was to determine whether acute ethanol exposure alters phosphorylation of conventional PKC isoforms at a threonine 674 (p‐cPKC) site in the hydrophobic domain of the kinase, which is required for its catalytic activity.Methods: Male rats were administered a dose range of ethanol (0, 0.5, 1, or 2 g/kg, intragastric) and brain tissue was removed 10 minutes later for evaluation of changes in p‐cPKC expression using immunohistochemistry and Western blot methods.Results: Immunohistochemical data show that the highest dose of ethanol (2 g/kg) rapidly increases p‐cPKC immunoreactivity specifically in the nucleus accumbens (core and shell), lateral septum, and hippocampus (CA3 and dentate gyrus). Western blot analysis further showed that ethanol (2 g/kg) increased p‐cPKC expression in the P2 membrane fraction of tissue from the nucleus accumbens and hippocampus. Although p‐cPKC was expressed in numerous other brain regions, including the caudate nucleus, amygdala, and cortex, no changes were observed in response to acute ethanol. Total PKCγ immunoreactivity was surveyed throughout the brain and showed no change following acute ethanol injection.Conclusions: These results suggest that ethanol rapidly promotes phosphorylation of cPKC in limbic brain regions, which may underlie effects of acute ethanol on the nervous system and behavior.