Novel therapies are clearly needed for gliomas, and the combination of oncolytic vectors with chemotherapy possesses a significant hope for the treatment of this malignancy. In addition, combination with chemotherapy allows for lower virus doses to achieve anticancer effect, thus resulting in lower undesirable toxicities due to viral proteins. In this work, we sought to determine whether combination of an oncolytic adenovirus ICOVIR-5, with RAD001 or temozolomide (TMZ) could result in enhanced anti-glioma effect in vivo. We assessed the in vitro cytotoxic effect and replication properties of ICOVIR-5 in combination with RAD001 or TMZ in U87 MG glioma cell line by MTT and TCID(50), respectively. Our data showed that in vitro treatment with RAD001 or TMZ not only interfered with adenovirus replication but, in addition, enhanced its oncolytic properties. To evaluate the in vivo anticancer effect, athymic mice bearing glioma xenografts (5 x 10(5) U87 MG cells/animal) received a single intratumoral injection of ICOVIR-5 (10(7) PFU/animal). RAD001 was given as a regimen of 5 mg/kg 5 days per week until the end of the experiment and TMZ was administered for 5 days at 7.5 mg/kg/mice. Of significance, combination of ICOVIR-5 with RAD001 or TMZ showed a potent anti-glioma effect in vivo, resulting in a dramatic extension of the median animal survival and in 20-40% animals becoming free of disease beyond 90 days.