ArgBP2 (Arg/Abl-Binding Protein) is expressed at high levels in the heart and is localized in the Z-bands of mature myofibrils. ArgBP2 is a member of a small family of proteins that also includes vinexin and CAP (c-Cbl-associated protein), all characterized by having one sorbin homology (SOHO) domain and three C-terminal SH3 domains. Antibodies directed against ArgBP2 also react with the Z-bodies of myofibril precursors: premyofibrils and nascent myofibrils. Expression in cardiomyocytes of plasmids encoding Yellow Fluorescent Protein (YFP) fused to either full length ArgBP2, the SOHO, mid-ArgBP or the SH3 domains of ArgBP2 led to Z-band targeting of the fusion proteins, whereas an N-terminal fragment lacking these domains did not target to Z-bands. Although ArgBP2 is not found in skeletal muscle cells, YFP-ArgBP2 did target to Z-bodies and Z-bands in cultured myotubes. GST-ArgBP2-SH3 bound actin, -actinin and vinculin proteins in blot overlays, cosedimentation assays, and EM negative staining techniques. Over-expression of ArgBP2 and ArgBP2-SH3 domains, but not YFP alone, led to loss of myofibrils in cardiomyocytes. Fluorescence recovery after photobleaching was used to measure the rapid dynamics of both the full length and some truncated versions of ArgBP2. Our results indicate that ArgBP2 may play an important role in the assembly and maintenance of myofibrils in cardiomyocytes. © 2010 Wiley-Liss, Inc.