Apolipoprotein (apo) M is a novel apolipoprotein belonging to the lipocalin protein superfamily, i.e. proteins binding small lipophilic compounds. Like other apolipoproteins, it is expressed in hepatocytes and secreted into plasma where it associates with high-density lipoprotein particles. In addition, apoM is expressed at high levels in the kidney tubule cells. In this study, we show that the multiligand receptor megalin, which is expressed in kidney proximal tubule cells, is a receptor for apoM and mediates its uptake in the kidney. To examine apoM binding to megalin, a recombinant apoM was expressed in Escherichia coli and used in surface plasmon resonance and cell culture studies. The results showed apoM binding to immobilized megalin [dissociation constant (Kd) approximately 0.3-1 microm] and that the apoM was endocytosed by cultured rat yolk sac cells in a megalin-dependent manner. To examine the importance of apoM binding by megalin in vivo, we analyzed mice with a tissue-specific deficiency of megalin in the kidney. Megalin deficiency was associated with pronounced urinary excretion of apoM, whereas apoM was not detected in normal mouse, human, or rat urine. Gel filtration analysis showed that the urinary apoM-containing particles were small and devoid of apoA-I. The results suggest that apoM binds to megalin and that megalin-mediated endocytosis in kidney proximal tubules prevents apoM excretion in the urine.