The type VI intermediate filament (IF) protein synemin is a very large, unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin and/or vimentin forming heteropolymeric intermediate filaments (IFs) within mammalian muscle cells. Previous studies in our lab showed that avian synemin interacts with vinculin and α-actinin, both of which are cytoskeletal proteins present in adhesion sites such as the focal adhesions within cells. Thus, synemin may link the heteropolymeric IFs to adhesion sites within mammalian muscle cells or within some non-muscle cells that express synemin. At least two isoforms of synemin, namely α- and β-synemin, are present in mammals. The larger α-synemin is identical with the smaller β-synemin, with the only exception being that the slightly smaller β-synemin lacks a 312 amino acid insert (SNTIII) near the end of the long C-terminal tail domain. Whether the two mammalian synemin isoforms have different functions is unclear. Studies in this dissertation provide evidence that the two large mammalian (human) synemin isoforms interact differentially with several cytoskeletal proteins and thereby may fulfill different cellular functions. Mapping the binding site(s) of human synemins within vinculin and talin by in vitro protein-protein interaction assays revealed that SNTIII, which is absent in β-synemin, interacts specifically with both vinculin and talin. Transient expression of enhanced green fluorescent protein (EGFP)-tagged SNTIII co-localized with vinculin and talin at focal adhesion sites within mammalian cells. Confocal microscope analysis of intracellular localizations of endogenous synemin and vinculin/talin indicated that synemin co-localized with vinculin and talin at the sites of costameres, which are considered specialized focal adhesions located periodically along and immediately subjacent to the sarcolemma of mammalian striated muscle cells. These results indicated that mammalian α-synemin, but not β-synemin, interacts directly with the cytoskeletal proteins vinculin and talin within mammalian cells. To elucidate the possible functions of, and to identify novel interacting proteins for, the smaller β-synemin within cells, yeast two-hybrid screening of a human adult skeletal muscle cDNA library was performed using the entire tail domain of human β-synemin (SNβT) as the bait. The LIM domain protein zyxin was identified as an interaction partner of β-synemin. The interaction was further confirmed by several in vitro protein-protein interaction assays. Furthermore, over-expression of the zyxin-binding region of synemin within mammalian cells blocked the localizations of endogenous zyxin to the focal adhesions without disrupting normal cellular architectures. Knockdown synemin expression within cells by siRNA resulted in significantly compromised cell adhesion and migration. These results in toto indicate that mammalian (human) synemin isoforms participate in the focal adhesion dynamics and are essential for cell adhesion and cell motility.