The Walker rat 256 carcinosarcoma is a spontaneous rat breast tumor which has two unique variants, one of which is always associated with severe hypercalcemia and one which is not associated with hypercalcemia when the tumor is carried im. To determine the factor responsible for hypercalcemia, we established tumor cells derived from both variants in culture and found that the tumor cells from the hypercalcemic variant release an acid-soluble and acid-stable bone-resorbing factor of approximately 25,000 mol wt. Cells from the normocalcemic variant produce no bone-resorbing activity. These data are consistent with the notion that hypercalcemia in this animal model of the humoral hypercalcemia of malignancy is due to a macromolecular bone-resorbing factor which is acid and heat stable and requires disulfide bonds for biological activity.