The discovery of the genes for nephrin and podocin, which are mutated in two types of congenital nephrotic syndrome, was pivotal in establishing the podocyte as the central component of the glomerular filtration barrier. In vivo the proteins have been localized to the podocyte slit diaphragm, and there is recent evidence for interaction between the two via the adapter molecule CD2AP. We describe in a human podocyte cell line, the subcellular distribution of nephrin, podocins, and CD2AP and their functional interaction with the cytoskeleton. In addition to membrane expression, nephrin and podocin were detected intracellularly in a filamentous pattern. Double immunolabeling and depolymerization studies showed that nephrin and podocin partially co-localize with actin, most strikingly seen protruding from the tips of actin filaments, and are dependent on intact actin polymers for their intracellular distribution. Treatment of differentiated podocytes with puromycin aminonucleoside, an agent that causes foot process effacement in vivo, disrupted actin and nephrin simultaneously, with loss of cell surface localization. We demonstrate an intimate relationship between nephrin podocin and filamentous actin, and reason that disruption of nephrin/podocin could be a final common pathway leading to foot process effacement in proteinuric diseases.