The circulating concentrations of 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D are abnormally low in patients with chronic renal failure (CRF). To determine the importance of substrate (25-hydroxyvitamin D) concentration in this phenomenon, five patients with end stage renal disease treated with hemodialysis were given 25-hydroxyvitamin D3 (25-OH-D3) orally for 4 weeks. The serum concentration of 25-OH-D3 increased from a mean (+/- SEM) of 26 +/- 5 ng/ml immediately before therapy to a maximum of 108 +/- 5 ng/ml 4 weeks after beginning administration of 25-OH-D3. The concentrations of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), and 25,26-dihydroxyvitamin D3 (25,26(OH)2D3) increased from 6.6 +/- 0.8 pg/ml, 0.29 +/- 0.10 ng/ml, and 0.36 +/- 0.06 ng/ml, respectively, immediately before 25-OH-D3 administration to 21.7 +/- 2.2 pg/ml, 0.48 +/- 0.09 ng/ml; and 0.78 +/- 0.12 ng/ml, respectively, after 4 weeks of administration of 25-OH-D3. These results suggest that substrate availability may be an important determinant of the circulating concentrations of these metabolites in patients with CRF. It seems possible that the therapeutic effects of 25-OH-D3 administration to the CRF patient may be mediated through the normal actions of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and perhaps other metabolites rather than through analog effects of 25-OH-D3.