We studied the effect of the absence of bile salts on the absorption of vitamin D metabolites in vivo, in the biliary duct-ligated rat. The mesenteric, lymphatic absorption of the metabolites studied (vitamin D3, 25-hydroxyvitamin D3, and 1,25-dihydroxyvitamin D3) was almost completely abolished in the experimental animals. These results differed significantly (P less than 0.001) from those of the control rats. The 1,25-dihydroxyvitamin D3 absorption into the portal vein system was unaffected by the lack of biliary salts. The absorption of 25-hydroxyvitamin D3 was decreased and that of vitamin D3 was negligible, under the same experimental conditions. These data show that the more polar vitamin D metabolites, like 1,25-dihydroxyvitamin D3 and to some extent 25-hydroxyvitamin D3, are absorbed directly into the portal blood without the involvement of bile salts and micelle formation. Thus, the use of polar vitamin D metabolites should be considered in correcting hypovitaminosis D and osteomalacia in cases of chronic biliary salt depletion.