The chemical synthesis of [3alpha-3H]vitamin D2 of high specific activity has been described. With the use of this radioactive material, the existence of a polar metabolite believed to be the active form of vitamin D2 in the rat and chick has been demonstrated. It has been isolated in pure form from an in vitro chick kidney mitochondrial system and identified as 1,25-dihydroxyvitamin D2 by means of mass spectrometry, ultraviolet absorption spectrophotometry, and specific derivative synthesis. Its antirachitic activity equals that of 1,25-dihydroxyvitamin D3 in the rat.