Acetylcholine binding to muscarinic acetylcholine receptors activates G-proteins, phospholipase C, and protein kinase C (PKC), which phosphorylates brain Na⁺ channels and reduces peak Na⁺ current in hippocampal neurons. Because multiple PKC isozymes with different regulatory properties are expressed in hippocampal neurons, we investigated which ones are responsible for mediating this effect. The diacylglycerol analog oleoylacetylglycerol (OAG) reduced the amplitude of Na⁺ current in dissociated mouse hippocampal neurons by 28.5 ± 5.3% (p < 0.01). The reduction of peak Na⁺ current was similar with Ca²⁺-free internal solution and in 92 nm internal Ca²⁺, suggesting that calcium-dependent, conventional PKC isozymes were unlikely to mediate this response. Gö6976, which inhibits conventional PKC isozymes, reduced the effect of PKC activators only slightly, whereas rottlerin, which inhibits PKCδ preferentially at 5 μm, had no effect. Ro-31-8425 (20 nm), which inhibits conventional PKC isozymes, did not reduce the response to OAG. However, higher concentrations of Ro-31-8425 (100 nm or 1 μm) that inhibit novel PKC isozymes effectively blocked OAG inhibition of Na⁺ current. Inclusion of a selective PKCϵ-anchoring inhibitor peptide (PKCϵ-I) in the recording pipette prevented the reduction of peak Na⁺ current by OAG, whereas an anchoring inhibitor peptide specific for PKCβ and an inactive scrambled PKCϵ-I peptide had no effect. In addition, OAG had no effect on Na⁺ current in hippocampal neurons from PKCϵ null mice. Overall, our data from four experimental approaches indicate that anchored PKCϵ is the isozyme responsible for PKC-mediated reduction of peak Na⁺ currents in mouse hippocampal neurons.