The coreceptor usage of HIV-1 envelope proteins (Env) is mainly dependent on a defined variable region within the V3-loop of Env. Thus, retroviral vectors derived from murine leukemia virus (MLV), which have been pseudotyped with HIV-1 envelope proteins holding different V3-loops, enable selective gene delivery into either CXCR4 or CCR5 positive cultured cells. Here, we tested the distribution of CD4/CCR5-tropic [MLV(HIV)]-pseudotype vectors in transgenic mice expressing CD4 and either CXCR4 or CCR5 of human origin. The specificity of gene transfer was analyzed by ex vivo transduction of spleen cells as well as after i.v. or i.p. injection of transgenic mice. Expression of the transferred marker gene EGFP and vector sequences could be detected exclusively in lymphocytes expressing (hu)CD4 and (hu)CCR5, whereas MLV vectors pseudotyped with the VSV-G envelope glycoprotein mediated gene transfer in mice of all genotypes investigated. These data demonstrated that cell-specific gene delivery via [MLV(HIV)]-pseudotyped vectors, as previously shown for cultured cells, is also achievable in vivo.