We have studied the intestinal absorption of physiological amounts of vitamin D and 25-hydroxyvitamin D [25(OH)D3] in vivo from jejunal sacs in rats with thoracic and bile duct cannulas. Under all test conditions, absorption of 25(OH)D was greater than absorption of vitamin D. The majority of absorbed vitamin D and 25(OH)D was transported from the intestine in portal blood rather than lymph. When the luminal fluid contained 2.5 mM oleic acid and monoolein, the presence of taurocholate did not affect total intestinal absorption of vitamin D or 25(OH)D but increased recovery of vitamin in lymph. When luminal fat content was increased to 10 mM oleic acid and monoolein, total absorption of both vitamin D and 25(OH)D was enhanced by taurocholate. No significant metabolism of vitamin D or 25(OH)D occurred during absorption and transport in lymph. Fifty-three percent of lymph vitamin D was found in the chylomicron fraction, compared with only 13% of 25(OH)D. Inhibition of chylomicron synthesis by cycloheximide decreased vitamin D absorption by 46% but diminished 25(OH)D absorption by only 30%. These differences in behavior of vitamin D and 25(OH)D during absorption may explain the superior absorption of 25(OH)D in patients with malabsorption.