The metabolism of [3H]-25-hydroxyvitamin D3(3H-25-OH-D3) to [3H]-1,25-dihydroxyvitamin D3 (3H-1,25-(OH)2D)3 and [3H]24, 325 dihydroxyvitamin D3 (3H-24,25-(OH)2D3) was studied in sheep fetuses and lambs. In vivo metabolism was observed by chromatography of extracts of plasma obtained 24 hr after direct iv injection of 3H-25-OH-D3 to the fetus and lamb. The renal enzymes 25-hydroxyvitamin D-1-hydroxylase and -24-hydroxylase were determined in vitro by quantitative assay. In fetal lambs, 3H-24,25-(OH)2D3 was consistently the only detectable dihydroxyvitamin D3 metabolite; this was confirmed by the in vitro studies in which 24-hydroxylase was the only detectable enzyme. Parathyroid hormone infusion to the fetus did not stimulate 3H-1,25-(OH)2D3 production. In normal lambs fed sheep's milk or cow's milk 3H-24,25-(OH)2D3 was the only metabolite of 3H-25-OH-D3 in vivo and only 24-hydroxylase activity was detected in vitro. In lambs fed a low-calcium, vitamin D-free formula, 24-hydroxylase activity remained measurable for at least 11 days of feeding, and by that time 1-OHase activity became evident in vitro. Appreciable 3H-1,25-(OH)2D3 was not observed in plasma until 16 days of feeding with the formula. After 18 days on low-calcium, vitamin D-free formula, 1-OHase was the only detectable 25-OH-D3 metabolizing enzyme. We also studied the rates at which 3H-25-OH-D3, 3H-24,25-(OH)2D3 and 3H-1,25-(OH)2D3 disappeared from the lamb plasma compartment. Within 1 hr, 3H-1,25-(OH)2D3 fell to 50% of its initial concentration, and after 72 hr only 2% was left. In the case of 3H-25-OH-D and 3H-24,25-(OH)2D3, 50% of each metabolite was left after 7 to 10 hr and about 20% after 72 hr. The rapid disappearance of 3H-1,25-(OH)2D3 may explain the low levels or absence of this metabolite observed in the plasma of some animals shown to have renal 1-hydroxylase activity.