The effects of 1α,25-dihydroxyvitamin D 3, 24 R,25-dihydroxyvitamin D 3 and 1α,24 R,25-trihydroxyvitamin D 3 on active calcium and phosphate transport by rat duodenum were studied in vitamin D-deficient rats that either underwent sham surgery or were bilaterally nephrectomized. Both 1α, 25-dihydroxy- and 1α,24 R,25-trihydroxyvitamin D 3 markedly stimulated calcium and phosphate absorption with similar effects in shamoperated and nephrectomized rats. A 10-fold higher dose of 24 R,25-dihydroxyvitamin D 3 was required for an equivalent stimulation of absorption in sham-operated rats, and this compound had no effect on duodena from nephrectomized rats. These data provide the first evidence that 24 R,25-dihydroxy- and 1α,24 R,25-trihydroxyvitamin D 3 can stimulate the active intestinal absorption of phosphate. The lack of response to 24 R,25-dihydroxyvitamin D 3 in nephrectomized rats confirms prior results which indicated that renal metabolism of this secosteroid to 1α,24,25-trihydroxyvitamin D 3 is required for biological activity. In addition, we describe a simple bioassay technique which apparently reflects, with reasonable accuracy, the changes in duodenal calcium and phosphate absorption which occur under more rigorous short-circuited conditions and, in particular, can be used for screening putative 1α-hydroxyl analogs of vitamin D in nephrectomized rats.