We examined ten cellular or tissue sources of lymphocytes for specific binding of 1,25(OH) 2D 3, the hormonally active form of vitamin D 3. A specific-binding protein was found in three of these sources. Scatchard analysis of cytosol from a follicular lymphoma cell line revealed binding sites with a Kd of 7.0 × 10 −11 and a receptor concentration of 6.6 fmol/mg protein. Sucrose density centrifugation of 3H-1,25(OH) 2D 3 labeled cytosol showed a 3.75 peak which was absent in cytosols incubated with excess nonradioactive 1,25(OH) 2D 3. The relative amounts of vitamin D 3 metabolites required to displace 50% of the specifically bound 3H-1,25(OH) 2D 3 were 1,25(OH) 2D 3: 1,24,25(OH) 3D 3: 25(OH)D 3: 24,25(OH) 2D 3 = 1: 180: 1000: 2700. Excess vitamin D 3, cortisol, and estradiol failed to displace 3H-1,25(OH) 2D 3. Scatchard analysis of spleen cytosol from a patient with prolymphocytic transformation of chronic lymphocytic leukemia demonstrated a binding protein with a Kd of 1.2 × 10 −10 and a receptor concentration of 0.2 fmol/mg protein. DNA cellulose binding confirmed the presence of the specific-binding protein in this cytosol. Specific binding of 3H-1,25(OH) 2D 3 was also quantitated in a cell line from a patient with Burkitt's lymphoma with a Kd of 0.3 × 10 −10 and a receptor concentration of 29.6 fmol/mg protein. No specific binding of 3H-1,25(OH) 2D 3 was observed in lymphocytes from seven other malignant and nonmalignant sources. These results are the first to demonstrate a specific-binding protein for 1,25(OH 2D 3 in lymphocytes from tissue and from these specific cell lines. The presence of this protein in some lymphocytes but not others may reflect the state of activation of the lymphocytes.