Disabled-2 (Dab2) is a cytoplasmic adaptor protein that binds to the cytoplasmic tail of the multiligand endocytic receptor megalin, abundantly expressed in renal proximal tubules. Deletion of Dab2 induces urinary increase in specific plasma proteins such as vitamin D binding protein and retinol binding protein (Morris et al. EMBO J., 21: 1555- 1564, 2002). However, the subcellular localization of Dab2 in the renal proximal tubule and its function has not been fully elucidated yet. Here we report the characterization of Dab2 in the renal proximal tubule. Immunohistocytochemistry revealed co-localization with megalin in coated- pits and vesicles, but not in dense apical tubules and the brush border. Kidney-specific megalin knock-out almost abolished Dab2 staining, indicating that Dab2 subcellular localization requires megalin in the proximal tubule. Reciprocally, knock-out of Dab2 led to a redistribution of megalin from endosomes to microvilli. In addition, there was an overall decrease in levels of megalin protein observed by immunoblotting, but no decrease in clathrin or -adaptin protein levels or in megalin mRNA. In rat yolk sac epithelial BN16 cells Dab2 was present apically and co-localized with megalin. Introduction of anti-Dab2 antibody into BN16 cells decreased the internalization of ¹²⁵I-RAP, substantiating the role of Dab2 in megalin-mediated endocytosis. The present study shows that Dab2 is localized in the apical endocytic apparatus of the renal proximal tubule and that this localization requires megalin. Furthermore, the study suggests that the urinary loss of megalin ligands observed in Dab2 knock-out mice is caused by suboptimal trafficking of megalin leading to decreased megalin levels.