We explored the involvement of protein kinase C (PKC) and its isoforms in the regulation of BNaC2. Reverse transcriptase PCR evaluation of PKC isoform expression at the level of mRNA revealed the presence of a and ε/ε′ in all glioma cell lines analyzed; most, but not all cell lines expressed δ and ζ. No messages were found for the βI and βII isotypes of PKC in the tumor cells. Normal astrocytes expressed β but not γ. The essential features of these results were confirmed at the protein level by Western analysis. This disproportionate pattern of PKC isoform expression in glioma cell lines was further echoed in the functional effects of these PKC isoforms on BNaC2 activity in bilayers. PKC holoenzyme or the combination of PKCβI and PKCβII isoforms inhibited BNaC2. Neither PKCε nor PKCζ or their combination had any effect on BNaC2 activity in bilayers. The inhibitory effect of the PKCβI and PKCβII mixture on BNaC2 activity was abolished by a 5-fold excess of a PKCε and PKCζ combination. PKC holoenzymes, PKCβI, PKCβII, PKCδ, PKCε, and PKCζ phosphorylated BNaC2 in vitro. In patch clamp experiments, the combination of PKCβI and PKCβII inhibited the basally activated inward Na+ conductance. The variable expression of the PKC isotypes and their functional antagonism in regulating BNaC2 activity support the idea that the participation of multiple PKC isotypes contributes to the overall activity of BNaC2.