Nephrotoxicity of myoglobin is well recognized as playing a part in the development of acute renal failure in settings of myoglobinuria. However, the molecular mechanism of myoglobin uptake in renal proximal tubules has not been clarified. Here, we report that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin. Both receptors were captured from solubilized renal brush-border membranes by affinity chromatography using myoglobin-Sepharose. Myoglobin bound to purified megalin and cubilin with Kd values of 2.0 and 3 microM, respectively, as evaluated by surface plasmon resonance analysis. Apomyoglobin bound to megalin with the same affinity, and the affinity of apomyoglobin to cubilin was reduced (Kd = 5 microM). Radioiodinated myoglobin could be displaced by apomyoglobin in inhibition studies using isolated renal brush-border membranes (Ki approximately 2 microM). Receptor-associated protein as well as antibodies directed against megalin and cubilin markedly inhibited the uptake of fluorescent-labeled myoglobin by cultured yolk sac BN-16 cells. The significance of megalin- and cubilin-mediated endocytosis for myoglobin uptake in vivo was demonstrated by use of kidney-specific megalin knockout mice. Injected myoglobin was extensively reabsorbed by megalin-expressing proximal tubular cells, whereas there was very little uptake in the megalin-deficient cells. In conclusion, this study establishes the molecular mechanism of myoglobin uptake in the renal proximal tubule involving the endocytic receptors megalin and cubilin. Identification of the receptors for tubular uptake of myoglobin may be essential for development of new therapeutic strategies for myoglobinuric acute renal failure.