Bisphosphonates (BPs) are effective inhibitors of tumor-induced bone resorption. Recent studies have demonstrated that BPs inhibit growth, attachment and invasion of cancer cells in culture and promote apoptosis. The mechanisms responsible for the observed anti-tumor effects of BPs are beginning to be elucidated. Recently, we reported that nitrogen-containing bisphosphonates (N-BPs) induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of farnesyl diphosphate synthase in the mevalonate pathway. Similar to AppCp-type metabolites of non-N-BPs, ApppI is able to induce apoptosis. This study investigated BP-induced ATP analog formation and its effect on cancer cell growth. To evaluate zoledronic acid (a N-BP)-induced ApppI accumulation, inhibition of protein prenylation and clodronate (a non-N-BP) metabolism to AppCCl2p, MCF-7 and MDA-MB-436 breast cancer cells, MCF-10A nonmalignant breast cells, PC-3 prostate cancer cells, MG-63 osteosarcoma cells, RPMI-8226, and NCI-H929 myeloma cells were treated with 25 micromol/l zoledronic acid or 500 micromol/l clodronate for 24 h. The inhibition of cell growth by zoledronic acid and clodronate was studied in MCF-7, MDA-MB-436, and RPMI-8226 cells by exposing the cells with 1-100 micromol/l zoledronic acid or 10-2000 micromol/l clodronate for 72 h. Marked differences in zoledronic acid-induced ApppI formation and clodronate metabolism between the cancer cell lines were observed. The production of cytotoxic ATP analogs in tumor cells after BP treatment is likely to depend on the activity of enzymes, such as farnesyl diphosphate synthase or aminoacyl-tRNA synthetases, responsible for ATP analog formation. Additionally, the potency of clodronate to inhibit cancer cell growth corresponds to ATP analog formation.