The spatial arrangement of the cell surface membranes (sarcolemma and transverse (T)-tubules) and internal membranes of the sarcoplasmic reticulum (SR) relative to the myofibril is critical for effective excitation-contraction (EC) coupling in cardiac myocytes, however the molecular determinants of this order remains to be defined. Here, we ascribe molecular and cellular properties to the coiled-coil, tail-anchored sarcolemmal membrane associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of the cardiomyocyte. The expression of SLMAP was developmentally regulated and its localization was distinctly apparent at the level of the membranes involved in regulating the E-C coupling mechanism. Several SLMAP isoforms were expressed in the cardiac myocyte with unique C-terminal membrane anchors which could target this molecule to distinct sub-cellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in cardiac muscle. The cardiac specific expression of SLMAP isoforms that can be targeted to distinct sub-cellular membranes, self assemble and interact with the myofibril suggest a potential role for this molecule in the structural arrangement of the E-C coupling apparatus.