To clarify in human beings the further metabolism of 25-OH-D3, the principal circulating form of vitamin D, the fates of 26,27-3H-25-OH-D3 or 1,2-3H-4-14C-D3 were compared in 6 normal subjects and 10 anephric patients on chronic hemodialysis. Normal subjects and 6 of 10 anephrics formed 24,25-(OH)2-D3 suggesting that the kidney may not be the only tissue capable of 24-hydroxylation. Normal subjects also produced metabolites co-migrating with authentic 1,25-(OH)2-D3 and 1,24-25-(OH)3-D3. Anephrics did not produce metabolites co-migrating with 1,25-(OH)2-D3 but their serum did contain small amounts of metabolites co-migrating with authentic 1,24,25-(OH)3-D3. Estimated plasma 25-OH-D3 pools were indistinguishable in the two groups averaging 91 ± 9 μg in normals and 96 ± 8 μg in anephrics. However, the estimated turnover rate of 25-OH-D3 in anephrics was only 1.6 ± 0.1 μg/day as compared to 2.8 ± 0.3 μg/day in normals (p < 0.005) confirming the importance of the human kidney in the further metabolism of vitamin D. Vitamin D and its metabolites were not dialyzable. Metabolites of 25-OH-D3 more polar than 1,24,25-(OH)3-D3 were also observed in plasma, urine, bile, and feces of both normals and anephrics. Their identity and biologic significance remain to be determined.