A major obstacle that limits the potential of human gene therapy is the inefficiency of gene delivery to appropriate sites in vivo. Previous studies demonstrated that the physiological surveillance function performed by von Willebrand factor (vWF) could be incorporated into retroviral vectors by molecular engineering of the MuLV ecotropic envelope (Env) protein. To advance the application of vWF targeting technology beyond laboratory animals, we prepared an extensive series of Env proteins bearing modified vWF-derived matrix-binding sequences and assembled these chimeric proteins into targeted vectors that are capable of transducing human cells. Initially, a dual envelope configuration was utilized, which required coexpression of a wild-type amphotropic Env. Subsequently, streamlined "escort" Env proteins were constructed wherein the inoperative receptor-binding domain of the targeting partner was replaced by the vWF-derived collagen-binding motif. Ultimately, an optimal construct was developed that exhibited properties of both extracellular matrix (ECM)-targeting and near wild-type amphotropic infectivity, and could be arrayed as a single envelope on a retroviral particle. On intraarterial instillation, enhanced focal transduction of neointimal cells (approximately 20%) was demonstrated in a rat model of balloon angioplasty. Moreover, transduction of tumor foci (approximately 1-3%) was detected after portal vein infusion of a matrix-targeted vector in a nude mouse model of liver metastasis. We conclude that the unique properties of these targeted injectable retroviral vectors would be suitable for improving therapeutic gene delivery in numerous clinical applications, including vascular restenosis, laser and other surgical procedures, orthopedic injuries, wound healing, ischemia, arthritis, inflammatory disease, and metastatic cancer.