Investigation of the role of individual protein kinase C (PKC) isozymes in the regulation of Na⁺ channels has been largely limited by the lack of isozyme-selective modulators. Here we used a novel peptide-specific activator (εV1–7) of εPKC and other peptide isozyme-specific inhibitors in addition to the general PKC activator phorbol 12-myristate 13-acetate (PMA) to dissect the role of individual PKCs in the regulation of the human cardiac Na⁺ channel hH1, heterologously expressed in Xenopus oocytes. Peptides were injected individually or in combination into the oocyte. Whole cell Na⁺ current (I _Na) was recorded using two-electrode voltage clamp. εV1–7 (100 nM) and PMA (100 nM) inhibited I _Na by 31 ± 5% and 44 ± 8% (at −20 mV), respectively. These effects were not seen with the scrambled peptide for εV1–7 (100 nM) or the PMA analog 4α-phorbol 12,13-didecanoate (100 nM). However, εV1–7- and PMA-induced I _Na inhibition was abolished by εV1–2, a peptide-specific antagonist of εPKC. Furthermore, PMA-induced I _Na inhibition was not altered by 100 nM peptide-specific inhibitors for α-, β-, δ-, or ηPKC. PMA and εV1–7 induced translocation of εPKC from soluble to particulate fraction in Xenopus oocytes. This translocation was antagonized by εV1–2. In native rat ventricular myocytes, PMA and εV1–7 also inhibited I _Na; this inhibition was antagonized by εV1–2. In conclusion, the results provide evidence for selective regulation of cardiac Na⁺channels by εPKC isozyme.