As an approach to cell targeting by retroviruses, the lack of which constitutes one major limitation of retroviral vector technology, we engineered the Moloney murine leukemia virus ecotropic envelope glycoprotein. When inserted between amino acids 6 and 7 of the latter, a single-chain antibody fragment (ScFv) specific for human major histocompatibility complex class I molecules was shown to be able to redefine the tropism of ecotropic Moloney murine leukemia virus-derived retroviral particles by allowing infection of major histocompatibility complex class I-positive human cells. At variance with other recently described experimental systems, the type of modification adopted here allowed targeted infection in the absence of coexpressed wild-type env-encoded protein molecules. Interestingly, the chimeric ScFv-env protein also retained the ability to recognize the ecotropic receptor and allowed infection of murine cells, albeit at a reduced efficiency.