Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of pro-tumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here I describe the development of two chimeric antigen receptors (CAR)s against the tumor vasculature, targeting either tumor endothelial marker 1 (TEM1) or prostate-specific membrane antigen (PSMA). CAR T cells incorporating scFv78, an scFv isolated against TEM1, were able to recognize immobilized plate-bound TEM1 protein, but were unable to recognize TEM1 on the surface of endothelial cell targets. In contrast, anti-PSMA CAR T cells, which incorporate the J591 scFv, were able to recognize human PSMA (hPSMA) both in vitro and in vivo. To elucidate the role of intracellular signaling domains on endothelial cell killing, a panel of the J591-based CAR T cells was characterized, each harboring a different combination of the intracellular signaling domains, CD3 zeta (z), CD28 (28), and CD137/4-1BB (BB). I found that all anti-hPSMA CAR T cells were able to recognize and eliminate PSMA+ endothelial targets in vitro, regardless of signaling domain. Furthermore, T cells bearing the 3rd generation anti-hPSMA CAR, P28BBz, were able to recognize and kill primary human endothelial cells isolated from gynecological cancers. In addition, the P28BBz CAR T cells were able to mediate regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine ovarian cancers models populated by murine vessels expressing hPSMA, the P28BBz CAR T cells were able to ablate PSMA+ vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA.