A renal ferredoxin was purified from bovine renal mitochondria to electrophoretic purity. The molecular weight of the renal ferredoxin was estimated by gel filtration and SDS-polyacrylamide gel electrophoresis to be 12,500 and 13,000, respectively. The optical absorption spectrum of renal ferredoxin in the oxidized form showed two peaks at 416 and 457 nm in the visible region, and the EPR absorption spectrum showed peaks at gx = gy =1.94 and gz = 2.02 in the reduced form at 13K. These spectra were typical of the 2S-2Fe type ferredoxins. Dissimilarities were recognized in the amino acid composition and isoelectric point between bovine renal ferredoxin and bovine adrenodoxin, but not in the optical, magnetic, and immunochemical properties. The reconstitution of 25-hydroxyvitamin D3-1 alpha-hydroxylase system was performed with the three components of NADPH-adrenodoxin reductase from bovine adrenal mitochondria, renal ferredoxin, and cytochrome P-450(1) alpha from bovine renal mitochondria. The results showed that the renal ferredoxin was essential for the 1 alpha-hydroxylase activity of 25-hydroxyvitamin D3.