C24-Hydroxylation was evaluated as a possible activation pathway for vitamin D2 and vitamin D3. Routine assays showed that 24-hydroxyvitamin D2 and 1,24-dihydroxyvitamin D2 could be detected in rats receiving physiological doses (100 IU/day) of vitamin D2; however, 24-hydroxyvitamin D3 could not be detected in rats receiving similar doses of vitamin D3. In rats, 24-hydroxyvitamin D2 was very similar to 25-hydroxyvitamin D2 at stimulating intestinal calcium transport and bone calcium resorption. The biological activity of 24-hydroxyvitamin D2 was eliminated by nephrectomy, suggesting that 24-hydroxyvitamin D2 must undergo 1 alpha-hydroxylation to be active at physiological doses. In vivo experiments suggested that when given individually to vitamin D deficient rats, 24-hydroxyvitamin D2, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 were 1 alpha-hydroxylated with the same efficiency. However, when presented simultaneously, 24-hydroxyvitamin D2 was less efficiently 1 alpha-hydroxylated than either 25-hydroxyvitamin D3 or 25-hydroxyvitamin D2. 1,24-Dihydroxyvitamin D2 was also approximately 2-fold less competitive than either 1,25-dihydroxyvitamin D2 or 1,25-dihydroxyvitamin D3 for binding sites on the bovine thymus 1,25-dihydroxyvitamin D receptor. These results demonstrate that 24-hydroxylation followed by 1 alpha-hydroxylation of vitamin D2 represents a minor activation pathway for vitamin D2 but not vitamin D3.