The 25-hydroxyvitamin D₃ [25 (OH)D₃] binding proteins from rat lymph and plasma were isolated and purified by sequential use of gel filtration, affinity and ion exchange chromatographies. The properties of both proteins were examined by high-performance liquid chromatography [HPLC], electrophoretic and immunological methods. The results were as follows. (1) On HPLC analysis using a gel permeation column, the proteins showed the same retention times. (2) On disc and sodium dodecyl sulfate disc gel electrophoresis, they showed the same isoelectric points. (4) On immunoelectrophoresis against rat whole antiserum obtained from rabbit, they showed immunological identity. These results strongly suggest that the 25 (OH)D₃ binding proteins from rat lymph and plasma are the same protein.