The PTEN gene, located on chromosome 10, is a phosphatase in the phosphatidylinositol 3'-kinase (PI3'K)-mediated signal transduction pathway. PTEN inhibits the activation of Akt, a serine-threonine kinase involved in proliferative metabolic and antiapoptotic pathways, and has tumor suppressive properties. We created a PTEN adenoviral vector, Ad-MMAC, to assess the role of PTEN in the treatment of bladder cancer. Direct injection of Ad-MMAC into established subcutaneous UM-UC-3 (PTEN deleted, upregulation of phosphorylated Akt) and UM-UC-6dox (wild-type PTEN, upregulation of phosphorylated Akt) tumors in nude mice resulted in PTEN expression, apoptosis, and significantly decreased growth compared to Ad-CTR- or Phosphate-buffered saline (PBS)-treated tumors. UM-UC-3 tumors completely disappeared in all of mice treated with Ad-MMAC, but PBS- and Ad-CTR-treated UM-UC-3 tumors continued to grow rapidly. UM-UC-14 tumors (wild-type PTEN) were transiently suppressed by Ad-MMAC. Downregulation of vascular endothelial growth factor and decreased microvessel density were seen in tumors treated with Ad-MMAC in vivo. Combination therapy with Ad-MMAC and doxorubicin improved the in vivo efficacy of PTEN gene therapy in the doxorubicin-resistant cell line UM-UC-6dox. Treatment with Ad-MMAC and doxorubicin completely eradicated established UM-UC-6dox tumors in three of 10 mice. UM-UC-14 tumors were transiently suppressed by this combined treatment. These data demonstrate that PTEN gene therapy can effectively treat bladder cancers that have genomic alterations in PTEN. Furthermore, tumors that exhibit drug resistance associated with expression of phosphorylated Akt can be effectively treated with PTEN gene therapy and chemotherapy.