The bioactivity of chemically synthesized 1 alpha,25-dihydroxyergocalciferol (1,25(OH)2D2) was investigated in rachitic chicks and in vitamin D-deficient rats. In prophylactic and in curative chick assays 1,25(OH)2D2 is about 10 times less active than 1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3). Since in the same bioassay vitamin D2 is more than 80 times less active than vitamin D3, discrimination against vitamin D2 in chickens must occur at two points, before and after the formation of 1,25(OH)2D2. Receptor binding studies revealed that the chick duodenal receptor binds 1,25(OH)2D2 with the same capacity as 1,25(OH)2D3. In rats 1,25(OH)2D2 proved to have the same antirachitic activity as 1,25(OH)2D3 and might become of therapeutic interest for application in man and domestic animals if the expectations of lower toxicity are confirmed.