The receptor-associated protein (RAP) has been identified as a chaperone regulating the expression and processing of the LDL receptor-related protein. RAP also binds to the related 600-kD multiligand endocytic receptor megalin expressed in many absorptive epithelia including renal proximal tubule. The present study examines the effect of RAP gene disruption on megalin expression and subcellular distribution in the proximal tubule as well as the effect on tubular protein reabsorption. It is shown that RAP is important for the normal expression and function of megalin. Megalin expression was reduced to approximately 23% estimated by immunoblotting and supported by immunocytochemistry and by the amount of megalin recovered by RAP affinity chromatography. Light- and electron microscope immunocytochemistry as well as analyses on separated membrane fractions showed significant changes in the subcellular distribution of megalin. A significant reduction in the normal brush border labeling was observed in association with increased labeling of rough endoplasmic reticulum and the smooth paramembranous endoplasmic reticulum along the basolateral membranes. RAP deficiency was associated with changes in urinary protein composition, enabling the identification of alpha-amylase as a new ligand for megalin. In addition, an increased excretion of vitamin D-binding protein, a recently identified ligand to megalin, was observed supporting changes in tubular protein reabsorption. The present data show that RAP is of crucial importance for normal processing and function of megalin, suggesting a chaperone-like function of this protein in the kidney proximal tubule.