The transport forms of vitamin D₃ and 25-hydroxyvitamin D₃ [25 (OH) D₃] in lymph were studied in rats with thoracic duct cannulas. Under in vitro and in vivo conditions, the majority of vitamin D₃ recovered from lymph was found in the chylomicrons and other lipoproteins fractions. On the other hand, the major fraction containing 25 (OH) D₃ corresponded to the albumin and α-globulin fractions. In order to confirm the presence of binding protein having affinity to 25 (OH) D₃ in lymph, the binding protein was isolated and purified by procedures of gel filtration, affinity and ion exchange chromatography from the incubated mixture of thoracic duct lymph with 25 (OH) D₃. The purified binding protein showed a single band of protein on disc and sodium dodecyl sulfate-disc gel electrophoreses. Furthermore, the protein also gave a single peak on highperformance liquid chromatography (HPLC) using a gel permeation column. The molecular weight of the protein was calculated from the results of HPLC as 57000, a value which is apparently different from that of lymph albumin. These results suggest that vitamin D₃ and 25 (OH) D₃ are mainly transported from the intestine to the liver via the lymph duct by different carrier mediators as lipoprotein complex and protein-bound forms, respectively, in rats.