Regulatory proteins on muscle filaments are substrates for protein kinase C (PKC) but mechanisms underlying activation and translocation of PKC to this non-membrane compartment are poorly understood. Here we demonstrate that the epsilon isoform of PKC (epsilon-PKC) activated by arachidonic acid (AA) binds reversibly to cardiac myofibrils with an EC(50) of 86 nM. Binding occurred near the Z-lines giving rise to a striated staining pattern. The delta isoform of PKC (delta-PKC) did not bind to cardiac myofibrils regardless of the activator used, and the alpha isoform (alpha-PKC) bound only under strong activating conditions. Three established PKC anchoring proteins, filamentous actin (F-actin), the LIM domain protein Cypher-1, and the coatamer protein beta'-COP were each tested for their involvement in cytoskeletal anchoring. F-actin bound epsilon-PKC selectively over delta-PKC and alpha-PKC, but this interaction was readily distinguishable from cardiac myofilament binding in two ways. First, the F-actin/epsilon-PKC interaction was independent of PKC activation, and second, the synthetic hexapeptide LKKQET derived from the C1 region of epsilon-PKC effectively blocked epsilon-PKC binding to F-actin, but was without effect on its binding to cardiac myofilaments. Involvement of Cypher-1 was ruled out on the basis of its absence from detergent-skinned myofibrils that bound epsilon-PKC, despite its presence in intact cardiac myocytes. The epsilon-PKC translocation inhibitor peptide EAVSLKPT reduced activated epsilon-PKC binding to cardiac myofibrils in a concentration dependent manner, suggesting that a RACK2 or a similar protein plays a role in epsilon-PKC anchoring in cardiac myofilaments.