Human trophoblastic cells (T3M-3) metabolized 25-hydroxyvitamin D3 into two more polar metabolites during in vitro incubations. A two-step high pressure liquid chromatography system revealed two unique elution positions of those trophoblastic cell-derived metabolites that exactly co-migrated with the elution positions of 5(Z)-19-nor-10-oxo-25-hydroxyvitamin D3 and 5(E)-19-nor-10-oxo-25-hydroxyvitamin D3, respectively. These unique metabolites did not bind specifically to a protein receptor for 1,25-dihydroxyvitamin D3.