A novel synthesis of a radioactive compound of 1α-hydroxyvitamin D₃ (1α OHD₃) (1) and its pharmacokinetics are described. Radioactive 1αOHD₃ tritiated at 22 and 23 positions ([22, 23-³H₄]1αOHD₃) (5) was prepared via key reactions of the reduction of acetylenic side chain in the ketone (12) with tritium gas in the presence of palladium-charcoal and the subsequent Wittig reaction with the A-ring synthon (16). [22, 23-³H₄]1αOHD₃ (5) showed high specific radioactivity (111.5 Ci/mmol) and was used successfully in pharmacokinetics studeies with rats. In the pharmacockinetics studies, the plasma concentration level of the active form of vitamin D₃, 1α, 25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃], after oral or intravenous administration of [22, 23-³H₄]1αOHD₃ (5), showed longer half-life, lower maximum concentration, and lower area under the curve than those after treatment of 1α, 25(OH)₂D³ tritiated at 26 and 27 positions (4). These results might suggest a beneficial therapeutic utility of 1αOHD₃ (1) over the treatment of 1α, 25(OH)₂D₃ (2).