Intermediate filaments (IFs) in cardiomyocytes consist primarily of desmin, surround myofibrils at Z disks, and transmit forces from the contracting myofilaments to the cell surface through costameres at the sarcolemma and desmosomes at intercalated disks. Synemin is a type IV IF protein that forms filaments with desmin and also binds α-actinin and vinculin. Here we examine the roles and expression of the α and β forms of synemin in developing rat cardiomyocytes. Quantitative PCR showed low levels of expression for both synemin mRNAs, which peaked at postnatal day 7. Synemin was concentrated at sites of cell–cell adhesion and at Z disks in neonatal cardiomyocytes. Overexpression of the individual isoforms showed that α-synemin preferentially localized to cell-cell junctions, whereas β-synemin was primarily at the level of Zdisks. An siRNA targeted to both synemin isoforms reduced protein expression in cardiomyocytes by 70% and resulted in a failure of desmin to align with Z disks and disrupted cell–cell junctions, with no effect on sarcomeric organization. Solubility assays showed that β-synemin was soluble and interacted with sarcomeric α-actinin by coimmunoprecipitation, while α-synemin and desmin were insoluble. We conclude that β-synemin mediates the association of desmin IFs with Z disks, whereas α-synemin stabilizes junctional complexes between cardiomyocytes.—Lund, L. M., Kerr, J. P., Lupinetti, J., Zhang, Y., Russell, M. A., Bloch, R. J., Bond, M. Synemin isoforms differentially organize cell junctions and desmin filaments in neonatal cardiomyocytes.