AbstractPalladin fulfils a crucial function as a molecular scaffold in organizing and stabilizing the actin cytoskeleton. At least four major palladin isoforms exist due to different promoter usage and alternative splicing: a 200‐kDa isoform, a 140‐kDa isoform, and two isoforms with a size of 90–92 kDa. Here, we describe their expression during mouse development and in adult tissues. The 200‐kDa isoform is predominantly expressed in developing heart and skeletal muscle. The 140‐kDa isoform is expressed in various mesenchymal tissues, and also represents the major isoform of the brain. The 90–92‐kDa isoforms are almost ubiquitously expressed with the highest levels in smooth muscle‐rich tissues. Immunohistochemical and immunofluorecence staining with an anti‐200‐kDa isoform‐specific antiserum localizes the large isoform to the Z‐discs of cardiac and skeletal muscle cells. Interestingly, the expression of this isoform is initiated and increasing during in vitro differentiation and fusion of C2C12 myoblasts, suggesting that the 200‐kDa palladin isoform may play a scaffolding role during sarcomeric organization. Developmental Dynamics 237:3342–3351, 2008. © 2008 Wiley‐Liss, Inc.