Humoral hypercalcemia of malignancy is a common but incompletely understood syndrome in humans. In an effort to define an animal model of this syndrome, we studied the transplantable Rice-500 Leydig cell tumor in male Fisher rats. Animals were studied 4-9, 10-12, and 13-14 days after tumor transplantation. By days 13-14, tumor-bearing animals were significantly hypercalcemic, hypercalciuric, and hyperphosphaturic compared to control animals. Fractional phosphorus excretion was elevated 4-fold in the tumor-bearing group despite hypophosphatemia. Mean nephrogenous cAMP in the tumor-bearing animals was 5 times the value in controls at days 13-14, while simultaneous immunoreactive PTH levels were undetectable. Plasma 1,25-dihydroxyvitamin D was significantly elevated in the tumor-bearing animals on day 14. Quantitative bone histomorphometry showed uncoupling of bone cell function in the tumor group, with marked suppression of bone formation, while indices of bone resorption were more than 2-fold elevated. Conditioned medium from tumor cells grown in culture consistently showed activity in a fetal bone-resorbing assay. This activity was heat stable and had an estimated mol wt of 30,000-50,000 daltons. Incubation of cells with indomethacin had no effect on bone-resorbing activity. These data indicate that the mediator in the model shares some of the actions of PTH, but is clearly distinct from native PTH. The findings exactly parallel those in human humoral hypercalcemia of malignancy, with the elevated 1,25-dihydroxyvitamin D values being the sole exception. The demonstration of in vitro bone-resorbing activity will aid in further characterization of the mediator.