Fabry disease is caused by deficiency of lysosomal a-galactosidase A (a-Gal A) resulting in cellular accumulation of globotriaosylceramide (Gb3) which often leads to end-stage renal failure. Gb3 is accumulated in all cells of the nephron. Therapy with recombinant a-Gal A reduces accumulation of Gb3 in different organs, including kidney. Here we investigate renal distribution of a-Gal A in kidneys from normal human and Fabry patients. Urine of Fabry patients was analyzed for protein excretion. Renal distribution of i.v. administered a-Gal A into normal and a-Gal A deficient mice was evaluated by immunohistochemistry and autoradiography and urinary excretion was determined. High expression of a-Gal A in normal kidney was seen in all tubular segments and in interstitial cells but not in glomeruli and endothelial cells. After infusion into mice or patients, enzyme was recovered in the urine. Recombinant enzyme was also found in kidneys of normal and a-Gal A KO mice and localized to endosomes and lysosomes in proximal tubules and to glomerular podocytes. BiaCore experiments revealed binding to the endocytic receptor megalin. The present experiments demonstrated the normal renal distribution of a-Gal A and showed that i.v. administered enzyme to some extent is filtered in glomeruli and taken up by podocytes and proximal tubule cells indicating the beneficial effect of enzyme replacement therapy for these cells.