Palladin is a recently described phosphoprotein that plays an important role in cell adhesion and motility. Previous studies have shown that palladin overexpression results in profound changes in actin organization in cultured cells. Palladin binds to the actin-associated proteins ␣-actinin, vasodilator-stimulated phosphoprotein, profilin, Eps8, and ezrin, suggesting that it may affect actin organization indirectly. To determine its molecular function in generating actin arrays, we purified palladin and asked if it is also capable of binding to F-actin directly. In cosedimentation and differential sedimentation assays, palladin was found to both bind and cross-link actin filaments. This bundling activity was confirmed by fluorescence and electron microscopy. Palladin fragments were then purified and used to determine the sequences necessary to bind and bundle F-actin. The Ig3 domain of palladin bound to F-actin, and a palladin fragment containing Ig3, Ig4, and the region linking these domains was identified as a fragment that was able to bundle F-actin. Because palladin has multiple Ig domains, and only one of them binds to F-actin, this suggests that different Ig domains may be specialized for distinct biological functions. In addition, our results suggest a potential role for palladin in generating specialized, actin-based cell morphologies via both direct actin cross-linking activity and indirect scaffolding activity.The actin cytoskeleton is a dynamic assembly that provides the cell with mechanical support and elasticity (1, 2), participates in cell locomotion through the formation and disassembly of protrusions (3), and provides a scaffold for the trafficking of cellular components (4, 5) and organization of signaling complexes (6). The organization of actin networks within the cell is tightly controlled by a variety of regulatory proteins that either cross-link actin filaments into robust bundles or regulate the assembly and disassembly of actin filaments by: 1) capping/ uncapping or severing the filament ends or 2) promoting the polymerization of actin at specific sites (7). To date, more than 23 classes of proteins have been shown to cross-link actin filaments into tight parallel bundles, loosely spaced bundles, or flexible networks (8). Actin cross-linking proteins such as filamin (9, 10), ␣-actinin (11), and fascin (12) play important roles in maintaining cell shape and allowing cells to move and adhere to a substrate. In addition, in differentiated cells in vivo, actin cross-linking proteins have a critical function in generating specialized actin-based structures such as sarcomeres, microvilli, and stereocilia (reviewed in Refs. 13 and 14). Thus, the extraordinary diversity of actin-binding proteins provides cells with a wide variety of molecular tools for constructing both dynamic and stable arrays of actin filaments.Palladin is a recently described phosphoprotein that is widely expressed in vertebrate cells and tissues (15-17). Palladin exists as three major isoforms, which display apparent molecul...