Prostate cancer (CaP) is the most frequently diagnosed cancer in men and the second leading cause of cancer related deaths in American men. In 2003, CaP was expected to account for 220,900 cancer diagnoses and 28,900 cancer deaths. Despite recent advances in the early detection and treatment of locally advanced prostate cancer, the prognosis for patients with the advanced form of prostate cancer is grave. The explosion of research in the genetic and molecular events paralleling the development of the lethal form of the disease has lead to the identification of multiple therapeutic targets. Targeting the molecular mechanisms underlying CaP development and metastasis offers a novel approach towards the development of effective therapeutic modalities. As part of our long term goal the major focus of the current study was to integrate real-time intravital imaging of prostate tumor dynamics along with retroviral and adenoviral gene transfer approach for maximizing the therapeutic efficacy and bystander effect of apoptosis inducers by exploiting VP22-mediated tumor vasculature specific targeting. We have used intravital imaging for the real-time monitoring of tumor progression, angiogenesis and adenoviral mediated gene transfer in athymic nude mouse models of prostate cancer. The human prostate cancer cell lines, LNCaP, C4-2 and PC-3 were transduced with an enhanced green fluorescent protein expressing, gibbon ape leukemia virus (GALV) envelope pseudotyped retroviral vector MFG-EGFP-GALV. Flow cytometry revealed that the retroviral transduction efficiency is cell line dependent and was in the order PC-3 (72.46%)>LNCaP (44.37%)>C4-2 (34.47%). A fluorescent prostate tumor model was developed by subcutaneous injection of PC-3 cells in six weeks old, uncastrated, male athymic nude mice. The tumor bearing mice were injected with Dextran-Texas Red dye and subjected to sequential intravital imaging on days 17 and 24 using a dual photon Bio-Rad MRC-1024 microscope. Higly fluorescent EGFP expressing tumor cells were observed along with neo-vascularization of the tumor. Infiltration of Dextran-Texas Red dye in between the EGFP expressing tumor cells was observed thereby indicating leaky tumor vasculature. We have used this technique for the real time intravital imaging of Herpes simplex virus tegument protein VP22 mediated intercellular trafficking dynamics and novel tumor vasculature specific targeting in androgen dependent and androgen independent prostate tumor models following intra-tumoral as well as systemic delivery. For the very first time, we would like to report VP22-mediated tumor vasculature specific targeting while sparing the normal vasculature associated with hepatic and muscular tissues. Our striking and novel findings suggest that hybrid VP22-apoptosis inducers might act as a double edged sword by maximizing apoptosis induction and the associated bystander effect on one hand and selectively targeting the tumor vasculature destruction thereby potentiating the anti-angiogenic effects.