Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that is a primary stimulant of the vascularization of solid tumors. VEGF production is induced by oncogenic gene mutations in the tumor cells and by hypoxic conditions inside the tumor mass. Hypoxia and the locally increased concentration of VEGF lead to an up-regulation of VEGF receptor expression on tumor endothelial cells. Therefore, in the tumor microenvironment, there is an up-regulation of both VEGF and its receptor, leading to a high concentration of occupied receptor on tumor vascular endothelium. The VEGF:receptor complex presents an attractive target for the specific delivery of drugs or other effectors to tumor endothelium. In the present study, several hybridomas that secrete monoclonal antibodies against the VEGF:receptor (Flk-1) complex or against VEGF itself have been raised. Three of the antibodies (3E7, GV39M, and 11B5) bind with high affinity to the VEGF:Flk-1 complex in ELISA and to tumor endothelium in frozen sections of human tumors, rodent tumors, and human tumor xenografts. 3E7 and GV39M localize selectively to tumor endothelium after i.v. injection into mice bearing human tumor xenografts. Additionally, one antibody (2C3) was raised that blocks the interaction between VEGF and KDR/Flk-1. 2C3 inhibits VEGF-mediated growth of endothelial cells in vitro and localizes strongly to connective tissue in tumors after injection into mice bearing human tumor xenografts. These findings suggest that 3E7, GV39M, and 2C3 are candidates for targeting and imaging the vasculature or connective tissue of tumors.