A method of clearly separating synthetic 24R,25-dihydroxyvitamin D₃ and 24S,25-dihydroxyvitamin D₃ as their Tris-trimethylsilyl ether derivatives has been devised using high pressure liquid chromatography on silica-gel columns. Using this technique, biologically prepared 24,25-dihydroxyvitamin D₃ has been shown to be entirely 24R,25-dihydroxyvitamin D₃. The 24R,25-dihydroxyvitamin D₃ is almost as active as 25-hydroxyvitamin D₃ in stimulating intestinal calcium transport, elevation of serum phosphorus and calcification of bone in rachitic rats. On the other hand, the 24S,25-dihydroxyvitamin D₃ has little or no activity in the cure of rickets in rats and the elevation of serum phosphorus of rachitic rats. It has some bone calcium mobilization activity, but approaches the activity of 24R,25-dihydroxyvitamin D₃ in the stimulation of intestinal calcium transport. Both the bone calcium mobilization response and the intestinal calcium transport response to the R and S isomers is eliminated by nephrectomy, which illustrates that both isomers undergo 1-hydroxylation. The biological activity profile of the biologically generated 24,25-dihydroxyvitamin D₃ is identical to that of 24R,25-dihydroxyvitamin D₃ and not that of 24S,25-dihydroxyvitamin D₃, which supports the conclusion that the natural compound is 24R,25-dihydroxyvitamin D₃.