CD2-associated protein (CD2AP) is an adapter protein associating with several membrane proteins, including nephrin, mutated in congenital nephrotic syndrome of the Finnish type, and polycystin-2, mutated in type 2 autosomal dominant polycystic kidney disease. Both proteins have critical roles in the maintenance of the integrity of the nephrons. Previous studies have suggested a role for CD2AP in the regulation of the organization of the actin cytoskeleton, but it has not been known whether the postulated association between CD2AP and actin is direct or mediated by other proteins. In this study, we address this question by using various cellular and biochemical approaches. We show that CD2AP and F-actin partially colocalize in cultured cells and that disruption of the actin cytoskeleton results in disorganization of endogenous CD2AP. Using cytoskeletal fractionation by differential centrifugation, we demonstrate that a proportion of CD2AP associates with the actin cytoskeleton. Furthermore, using pure actin and purified CD2AP fusion proteins in an F-actin coprecipitation assay, we show that CD2AP directly associates with filamentous actin and that this interaction is mediated by means of the COOH terminus of CD2AP. The present results suggest that CD2AP is involved in the regulation of the actin cytoskeleton and indicate that CD2AP may act as a direct adapter between the actin cytoskeleton and cell membrane proteins, such as nephrin and polycystin-2. Alterations in these interactions could explain some of the pathophysiological changes in congenital nephrotic syndrome and polycystic kidney disease.