Several hydroxy vitamin D3 (OH-D3) derivatives were tested for biological activity by measuring 45Ca release from prelabeled rat fetal bones, in vitro. In a 72 h continuous culture, 1.alpha.,25-(OH)2-D3 produced a significant effect at 10 pg/ml. A similar effect was produced by 50 ng/ml of either 25-OH-D3 or 5,6-trans-25-OH-D3 and by 500 pg of 3 deoxy-1.alpha.,25-(OH)2-D3. The activity of 24R,25-(OH)2-D3 was greater than 24S,25-(OH)2-D3 and the R isomer had activity that more closely resembled the biosynthetic compound. At a concentration up to 1 .mu.g/ml 3-deoxy-1.alpha.-OH-D3 was inactive. Using a 24 h preincubation period with no added vitamin D3 derivative, a steep dose-response curve was obtained with 1.alpha.,25-(OH)2-D3 over a range of 2-10 pg/ml during a subsequent 96 h incubation period and 1.alpha.,25-(OH)2-D3 had about 5000 times the activity of 25-OH-D3.