Since our original demonstration of the metabolism of 1α,25(OH)2D3 into 1α,25(OH)2-3-epi-D3 in human keratinocytes, there have been several reports indicating that epimerization of the 3 hydroxyl group of vitamin D compounds is a common metabolic process. Recent studies reported the metabolism of 25OHD3 and 24(R),25(OH)2D3 into their respective C-3 epimers, indicating that the presence of 1α hydroxyl group is not necessary for the 3-epimerization of vitamin D compounds. To determine whether the presence of a 25 hydroxyl group is required for 3-epimerization of vitamin D compounds, we investigated the metabolism of 1αOHD3, a non-25 hydroxylated vitamin D compound, in rat osteosarcoma cells (ROS 17/2.8). We noted metabolism of 1αOHD3 into a less polar metabolite which was unequivocally identified as 1αOH-3-epi-D3 using the techniques of HPLC, GC/MS, and 1H-NMR analysis. We also identified 1αOH-3-epi-D3 as a circulating metabolite in rats treated with pharmacological concentrations of 1αOHD3. Thus, these results indicated that the presence of a 25 hydroxyl group is not required for 3-epimerization of vitamin D compounds. Furthermore, the results from the same studies also provided evidence to indicate that 1αOH-3-epi-D3, like 1αOHD3, is hydroxylated at C-25. We then evaluated the biological activities of 1αOH-3-epi-D3. Treatment of normal rats every other day for 7 days with 2.5 nmol/kg of 1αOH-3-epi-D3 did not raise serum calcium, while the same dose of 1αOHD3 increased serum calcium by 3.39 ± 0.52 mg/dl. Interestingly, in the same rats which received 1αOH-3-epi-D3 we also noted a reduction in circulating PTH levels by 65 ± 7%. This ability of 1αOH-3-epi-D3 to suppress PTH levels in normal rats without altering serum calcium was further tested in rats with reduced renal function. The results indicated that the ED50 of 1αOH-3-epi-D3 for suppression of PTH was only slightly higher than that of 1α,25(OH)2D3, but that the threshold dose of the development of hypercalcemia (total serum Ca > 10.5 mg/dl) was nearly 80 times higher. These findings indicate that 1αOH-3-epi-D3 is a highly selective vitamin D analog with tremendous potential for treatment of secondary hyperparathyroidism in chronic renal failure patients. © 2005 Wiley-Liss, Inc.