The Z-line represents a critical link between the transverse tubule network and cytoskeleton of cardiac cells with a role in anchoring structural proteins, ion channels, and signaling molecules. Protein kinase C-epsilon (PKC-) regulates cardiac excitability, cardioprotection and growth, possibly as a consequence of translocation to the Z-line/T-tubule region. To investigate the mechanism of PKC- translocation, fragments of its N-terminal 144 amino acid variable domain, V1, were fused with Green Fluorescent Protein (GFP) and evaluated by quantitative Fourier image analysis of decorated myocytes. Deletion of 23 amino acids from the N-terminus of V1, including an EAVSLKPT motif important for binding to a Receptor for Activated C Kinase (RACK2), reduced but did not abolish Z-line binding. Further deletions of up to 84 amino acids from the N-terminus of V1 also did not prevent Z-line binding. However, deletions of residues 85-144 from the C-terminus of V1 strongly reduced Z-line binding. C-terminal deletions resulted in a 2.5-fold greater loss of binding energy(G) than did N-terminal deletions. Synthetic peptides derived from these regions modulated V1 binding and cardiac myocyte function, but also revealed considerable heterogeneity within populations of adult cardiac myocytes. The C-terminal subdomain important for Z-line anchoring maps to a surface in the crystal structure of V1 that complements the 8 amino acid RACK2 binding site and two previously identified membrane docking motifs. PKC-anchoring at the cardiac Z-line/T-tubule appears to rely on multiple points of contact probably involving protein-lipid and protein-protein interactions.