Tumor angiogenesis is a rate-limiting factor for tumor growth, and the endothelial cells of tumor vessels display specific features that can be exploited for the selective delivery of cancer therapeutics. To specifically target exogenous genes to angiogenic tumor vessels, we generated a panel of vesicular stomatitis virus-pseudotyped lentiviral vectors (LVs) engineered for endothelial cell (EC)-specific expression. We cloned a wide repertoire of transcription regulatory sequences from genes preferentially expressed in ECs (Tie1, Tie2, Flk-1, VE-Cad, and ICAM-2) into self-inactivating LVs to drive expression of the marker gene encoding green fluorescent protein (GFP) or of the conditionally toxic gene encoding nitroreductase, and compared them with the ubiquitously expressing phosphoglycerate kinase (PGK) and cytomegalovirus (CMV) promoters. We evaluated the efficiency and specificity of vector expression in vitro in a panel of human primary cultures, including ECs, fibroblasts, neurons, lymphocytes, and hematopoietic progenitors, and in tumor cell lines. We found that vectors containing promoter and enhancer sequences from the Tie2 gene achieved remarkable specificity of expression in ECs in vitro and in vivo. On intravenous delivery into tumor-bearing mice, the Tie2 vector targeted expression to the ECs of tumor vessels. In contrast, LVs carrying the PGK or CMV promoter gave widespread GFP marking in ECs and non-ECs of tumors and other organs. The previously reported upregulation of the Tie2 gene in ECs activated for angiogenesis may explain the remarkable selectivity of expression of the Tie2 vector in ECs of tumor vessels. The new vector provides the means for selective delivery of gene therapy to tumor sites in vivo.