Bisphosphonates are widely clinically used inhibitors of bone resorption. Pre-clinical studies indicate that bisphosphonates also inhibit the growth of various cancer cells in vitro, but their in vivo anti-cancer activity varies greatly, depending on the tumor type. We compared the various cellular effects of bisphosphonates in breast cancer and mesothelioma cells, with differences in growth inhibition responses to bisphosphonate-treatment in vivo. We show that the growth inhibitory effects of nitrogen-containing bisphosphonates are significantly affected by excess Ca(2+) in a cell- and bisphosphonate-specific fashion. Furthermore, excess pyrophosphate-resembling bisphosphonates prevent nitrogen-containing-bisphosphonate-induced accumulation of unprenylated Rap1A, p38 phosphorylation and growth inhibition in human MDA-MB-231 breast cancer and mouse AB-12 mesothelioma cells. For some, but not all tested, pyrophosphate-resembling bisphosphonate: nitrogen-containing bisphosphonate combinations these results may be partially explained by the ability of the excess pyrophosphate-resembling bisphosphonates to chelate Ca(2+). In mice, subcutaneous AB-12 and MDA-MB-231 tumors exhibit positive staining for Ca(2+) minerals, as revealed with Von Kossa stainings. We further show that the AB-12 tumors accumulate significantly more of the bone scanning bisphosphonate, Tc99m-medronate, as compared with MDA-MB-231 tumors. In conclusion, our results suggest that Ca(2+) regulates the growth inhibitory effects of bisphosphonates in a target cell and drug-specific fashion. These findings may be of physiological relevance since many tumor types are calcified. They further suggest that bisphosphonates can accumulate in tumors that are growing at the visceral sites and that differences in tumor accumulation of bisphosphonates may regulate their in vivo sensitivity to these drugs.