Although many patients with humoral hypercalcemia of malignancy exhibit reduced serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels, N-terminal fragments of recently identified PTH-related protein as well as PTH itself elevate serum 1,25-(OH)2D concentrations. In the present study, the effect of tumor extracts from human tumor-implanted hypercalcemic nude rat models with high and low serum 1,25-(OH)2D on renal 1,25-(OH)2D3 production was examined using rat kidney cells in culture. Whereas tumors from rats with high serum 1,25-(OH)2D levels (OCC rats) contained only a single peak of cAMP production-stimulating activity (CPSA) in osteogenic sarcoma cells on reverse phase HPLC, tumor extracts from rats with low serum 1,25-(OH)2D levels (UCC rats) contained at least two peaks of CPSA. The main peak (peak A) was estimated to be approximately 17K by gel permeation chromatography, which was the same as the molecular size of the hitherto identified PTH-related protein, and a minor peak of CPSA (peak B) was estimated to be about 25K. When peak A or crude extracts of OCC tumors as well as human PTH-(1-34) were added to primary cultures of rat kidney cells, the production of 1,25-(OH)2D3 was significantly stimulated. In contrast, although peak B or crude UCC tumor extracts had no effect on 1,25-(OH)2D3 production in themselves, when they were added together with peak A or human PTH-(1-34) the stimulation of 1,25-(OH)2D3 production was almost completely inhibited. Both peak A and peak B enhanced cAMP production in cultured kidney cells, and the cAMP production by peak A was not affected by peak B. These results are consistent with the possibility that elaboration of an additional factor from tumor cells may be the mechanism by which serum 1,25-(OH)2D levels are suppressed in patients with humoral hypercalcemia of malignancy. The nature as well as the mechanism of action of this factor remain to be elucidated.