Recently, it was revealed that 1alpha,25-dihydroxyvitamin D3 (1alpha,25(
OH)2D3) and 24R,25-dihydroxyvitamin D3 (24,25(
OH)2D3) were metabolized to their respective epimers of the
hydroxyl group at C-3 of the A-ring. We now report the isolation and structural assignment of 3-epi-25-hydroxyvitamin D3 (3-epi-25(
OH)D3 as a major metabolite of
25-hydroxyvitamin D3 (25(
OH)D3) and the further metabolism of C-3 epimers of
vitamin D3 metabolites. When 25(
OH)D3 was incubated with various cultured cells including
osteosarcoma,
colon adenocarcinoma, and
hepatoblastoma cell lines, 3-epi-25(
OH)D3 and 24,25 (
OH)2D3 were commonly observed as a major and minor metabolite of 25(
OH)D3, respectively. 25(
OH)D3 was at least as sensitive to C-3 epimerization as 1alpha, 25(
OH)2D3 which has been reported as a substrate for the C-3 epimerization reaction. Unlike these cultured cells, LLC-PK1 cells, a porcine kidney cell line, preferentially produced 24,25(
OH)2D3 rather than 3-epi-25(
OH)D3. We also confirmed the existence of 3-epi-25(
OH)D3 in the serum of rats intravenously given pharmacological doses of 25(
OH)D3. The cultured cells metabolized 3-epi-25OHD3 and 3-epi-1alpha,25(
OH)2D3 to 3-epi-24,25(
OH)2D3 and 3-epi-1alpha,24,25(
OH)3D3, respectively. In addition, we demonstrated that 3-epi-25(
OH)D3 was metabolized to 3-epi-1alpha,25(
OH)2D3 by
CYP27B1 and to 3-epi-24,25(
OH)2D3 by CYP24 using recombinant Escherichia coli cell systems. 3-Epi-25(
OH)D3, 3-epi-1alpha,25(
OH)2D3, and 3-epi-24,25(
OH)2D3 were biologically less active than 25(
OH)D3, 1alpha,25(
OH)2D3, and 24,25(
OH)2D3, but 3-epi-1alpha,25(
OH)2D3 showed to some extent transcriptional activity toward target genes and anti-proliferative/differentiation-inducing activity against human
myeloid leukemia cells (HL-60). These results indicate that C-3 epimerization may be a common metabolic pathway for the major metabolites of
vitamin D3.