Palladin, a cytoskeletal protein with essential functions for stress fiber formation, is found in developing and mature tissues, including the kidney. To define its role in the kidney, we measured its expression in mouse kidney and found it co-localized with F-actin in smooth muscle cells of renal arterial vessels, mesangial cells, and podocytes but not in tubular epithelium. Using immunoelectron microscopy, we confirmed that palladin was present in podocytes. In cultured mouse podocytes, palladin co-localized with F-actin in dense regions of stress fibers, focal adhesions, cell-cell contacts and motile cell margins. Transfection with the N-terminal half of palladin targeted it to F-actin-containing structures in podocytes while the C-terminal half accumulated in the nucleus, a result also found for endogenous palladin in cultured cells after leptomycin B was used to block nuclear export. Green fluorescent protein (GFP)-tagged palladin was found in dynamic ring-like F-actin structures and ruffles in cultured podocytes after stimulation with epidermal growth factor. Inhibition of palladin expression by transfection of an antisense construct reduced the formation of ring-like structures. Photo-bleaching analysis showed that GFP-palladin turned over with a half-time of 10 s in focal adhesions and dense regions of stress fibers, suggesting that palladin is a dynamic scaffolding protein. Our study shows that palladin is expressed in podocytes and plays an important role in actin dynamics.