The cytoskeleton plays a profound role in striated muscle. Cytoskeletal defects in the intermediate filaments result in mild to moderate myopathies. My collaborators and I have shown that the ablation of two intermediate filaments, desmin and keratin 19, causes defects in sarcolemmal organization, mitochondrial distribution, and muscle performance. To better understand how these defects arise, I examined synemin, an intermediate filament that interacts with desmin and keratins and that localizes protein kinase A to subcellular structures. Two isoforms, alpha and beta, are expressed in striated muscle and each isoform has different localizations and potential binding partners. beta-Synemin was required for the association of desmin with the developing Z-disk; its interaction with desmin is required for synemin to concentrate at the Z-disk and the sarcolemma in mature muscle. By contrast, alpha-synemin was required for the stabilization of the developing intercalated disk in cardiomyocytes. In mature muscle, I found that the absence of keratin 19 resulted in the specific loss of alpha synemin from M-bands. Consistent with this, I identified a novel interaction of alpha-synemin with the giant muscle protein, titin, to a site on titin located at the M-band which may regulate the organization of keratin filaments there. As the intermediate filament network is closely associated with the microtubule network, I also examined the structure and stability of microtubules in muscle fibers lacking desmin and keratin. The absence of keratins decreased the stability of the microtubules, while the absence of desmin caused an increase in tubulin expression. The absence of either filament resulted in a drastic reorganization of sarcolemmal microtubules. These results extend the roles of desmin and keratin 19 filaments in striated muscle, and indicate that they act in part through synemin and the regulation of microtubules.