[6-3H]-1alpha-Hydroxyvitamin D3 was chemically synthesized and its full biological activity and radiochemical purity were demonstrated. With the use of this preparation it has been possible to demonstrate in vivo that in rats the [6-3H]-1Alpha-hydroxyvitamin D3 is converted to [6-3H]-1alpha,25-dihydroxyvitamin D3, the natural hormone. In fact, in the intestine and bone of rats given 32 picomoles of [6-3H]-1alpha-hydroxyvitamin D3 each day for 6 days, more than 80 percent of the lipid-soluble radioactivity exists as [6-3H]-1alpha,25-dihydroxyvitamin D3, a finding that suggests that much of the biological effectiveness of 1alpha-hydroxyvitamin D3 is due to its conversion to 1alpha,25-dihydroxyvitamin D3.