Hematopoietic cells are important targets for genetic modification with retroviral vectors. Attempts at human gene therapy of stem cells have achieved limited success partly because of low gene transfer efficiency. Chymotryptic fragments of the extracellular matrix molecule fibronectin used during infection have been shown to increase transduction of human hematopoietic progenitor cells. Here, we demonstrate that this enhanced gene transfer into mammalian target cells is due to direct binding of retroviral particles to sequences within the fibronectin molecule. Transduction of mammalian cells, including murine long-term repopulating hematopoietic cells, is greatly enhanced when cells are adherent to chimeric fragments containing these retroviral binding sequences. In addition, colocalization of retrovirus and target cells on fibronectin peptides allows targeted transduction of specific cell types by exploiting unique ligand/receptor interactions.