Cell surface antigens of transformed cells are the traditional targets for antibody-guided detection and therapy of solid neoplasms. Alternative targets may be found in the tumor stroma, which contains newly formed blood vessels, reactive fibroblasts, and extracellular matrix proteins. The F19 cell surface glycoprotein of reactive fibroblasts is a prototypic stromal antigen since it is found in the stroma of > 90% of common epithelial cancers but is absent or expressed at low levels in normal tissues and benign epithelial tumors. In the present study, we define an additional tumor stromal antigen, FB5, that is selectively expressed in vascular endothelial cells of malignant tumors. Immunohistochemical analysis of 128 tumors identified FB5 in endothelial cells in 67% of the samples (including 41 of 61 sarcomas, 26 of 37 carcinomas, and 18 of 25 neuroectodermal tumors) whereas normal blood vessels and other adult tissues tested lacked FB5 expression. In vitro studies showed that FB5 is a M(r) 165,000 cell surface glycoprotein, comprised of a M(r) 95,000 core polypeptide and highly sialyated O-linked oligosaccharides but few if any N-linked sugars, and that the FB5 gene is located on chromosome 11q13-qter. The restricted tissue distribution of the FB5 protein, which we refer to as endosialin, suggests strategies for tumor imaging and therapy that are aimed primarily at the tumor vasculature.