We are interested in developing oncolytic adenoviruses for the treatment of bone metastasis of cancer. A key limitation of systemic delivery of oncolytic adenovirus type 5 (Ad5) is that the majority of the virus is taken up by the liver, causing liver damage and systemic toxicity. Given that Ad5 hexon binding with blood coagulation factor X is a key factor in liver sequestration, and that a rare serotype, Ad48, has a diminished capacity to bind with factor X, we have generated mHAd.luc2, a novel hexon-chimeric oncolytic adenovirus. To create mHAd.luc2, seven hypervariable regions of Ad5 hexon were substituted with the corresponding regions from Ad48. Compared with Ad5-based oncolytic virus Ad.luc2, intravenous injection of mHAd.luc2 into nude mice resulted in significantly reduced liver uptake. A single high dose (1.0×10(11) viral particles/mouse) of Ad.luc2 resulted in 100% animal death by day 3; whereas none of the mice died in the mHAd.luc2 group. Liver enzyme and liver pathology studies indicated that mHAd.luc2 induced significantly less liver toxicity compared with Ad.luc2. Both mHAd.luc2 and Ad.luc2 exhibited similar binding with breast tumor cells, whereas in the presence of factor X, mHAd.luc2 binding was reduced. Both mHAd.luc2 and Ad.luc2 had nearly equal replication potential in breast cancer cells in vitro. Intravenous injection of mHAd.luc2 and Ad.luc2 into nude mice bearing bone metastases resulted in uptake of the viruses into skeletal tumors, and induced significant inhibition of established bone metastases. Thus, liver-detargeted oncolytic adenovirus can be developed for the treatment of breast cancer bone metastasis.