The Walker carcinosarcoma (WCS) 256 is a well-characterized rat model of humoral hypercalcemia of malignancy (HHM). We addressed the question of whether parathyroid hormone-related protein (PTHrP) is the factor responsible for mediating HHM in this model. WCS 256 cells were subcutaneously implanted in female rats. We examined the plasma at days 0, 2, 4, 6, and 8. The midregional PTHrP measured by radioimmunoassay (RIA) and the plasma calcium increased significantly. Measuring PTHrP by a two-site immunoradiometric assay (IRMA) showed comparable results. There was a strong positive correlation between plasma calcium and midregional PTHrP (r = 0.85, p < 0.0001). A strong positive correlation between tumor weight and both midregional PTHrP (r = 0.83, p < 0.0001) and plasma calcium (r = 0.87, p < 0.0001) was also found. After surgical removal of the tumor at day 5, both plasma calcium and plasma PTHrP levels fell to within the normal range. Ip administration of native polyclonal antiserum against PTHrP(53-84) led to a significant decrease of plasma calcium. Extracted WCS 256 tumor showed 5-fold increased levels of midregional PTHrP compared with liver. Immunohistochemistry and Western blot were positive for PTHrP. RNA from the WCS 256 tumor was positive for PTHrP whereas liver tissue RNA was negative. WCS 256 cells grown in vitro also secreted PTHrP into the medium. We conclude that PTHrP is synthesized and secreted by WCS 256 and that PTHrP is the factor responsible for mediating hypercalcemia in the WCS 256 rat model.