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This paper is the first definitive report demonstrating a unique membrane receptor for 1,25-dihydroxyvitamin D3(1,25(OH)2D3) which mediates the rapid and nongenomic regulation of protein kinase C (PKC). Previous studies have shown that 1,25(OH)2D3 exerts rapid effects on chondrocyte membranes which are cell maturation-specific, do not require new gene expression, and do not appear to act via the traditional vitamin D receptor. We used antiserum generated to a [3H]1,25(OH)2D3 binding protein isolated from the basal lateral membrane of chick intestinal epithelium(Ab99) to determine if rat costochondral resting zone (RC) or growth zone (GC) cartilage cells contain a similar protein and if cell maturation-dependent differences exist. Immunohistochemistry demonstrated that both RC and GC cells express the protein, but levels are highest in GC. The binding protein is present in both plasma membranes and matrix vesicles and has a molecular weight of 66,000 Da. The 66 kDa protein in GC matrix vesicles has a Kd of 17.2 fmol/ml and Bmax of 124 fmol/mg of protein for [3H]1,25(OH)2D3. In contrast, the 66 kDa protein in RC matrix vesicles has a Kd of 27.7 fmol/ml and a Bmax of 100 fmol/mg of protein. Ab99 blocks the 1,25(OH)2D3-dependent increase in PKC activity in GC chondrocytes, indicating that the 1,25(OH)2D3-binding protein is indeed a receptor, linking ligand recognition to biologic function.