Vitamin D-binding protein (DBP) is an abundant plasma protein. The observation of immunodetectable, cellassociated DBP on peripheral blood mononuclear cells and placental cytotrophoblasts had presented the question of the origin, function, and precise subcellular localization of cellassociated DBP. Using anti-human DBP F(ab’)2 with fluorescence- activated cytometric analysis and immunogold electron microscopy, we detected DBP on the plasmalemma of U937 cells, a monoblastic, histiocytic cell line grown in media supplemented with fetal calf serum (FCS). DBP was then removed from FCS by actin affinity chromatography followed by anti- DBP immunoaffinity chromatography. U937 cells in this DBPfree medium exhibited nearly identical growth rates to cells grown in medium containing native FCS. However, in contrast to cells grown with native FCS, those grown for seven to eight generations with DBP-free FCS exhibited less cell-surface DBP as quantified by fluorescence-activated cytometric analysis (73% decrease) and immunoelectron microscopy (88% decrease). DBP mRNA could not be detected in U937 cells, placental tissues, freshly prepared resting and stimulated B and T lymphocytes, or lymphocyte-derived cell lines by Northern analysis. In addition, using the sensitive reverse transcriptase/polymerase chain reaction assay no DBP fragments were detectable in U937 cells. We conclude that U937 cell-associated DBP is exogenously derived from plasma and is located on the plasmalemma. Based upon this conclusion, we postulate that specific binding sites for DBP may exist on the plasma membranes of certain cell types. (Endocrinology127: 2313–2321, 1990)