Retroviral vectors derived from murine leukemia virus (MLV) have been pseudotyped with a variant of the envelope glycoprotein (Env) of nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm) to result in [MLV(SIVagm-wt)] vector particles. The variant env gene encodes a full-length surface envelope glycoprotein (SU) and a C-terminally truncated transmembrane protein (TM). To change the coreceptor usage of this vector from CCR5 to CXCR4, which is predominant on human CD4-positive lymphocytes, the putative V3-loop of SIVagm SU was replaced by that of the T cell tropic HIV-1 variant BH10. The resulting [MLV(SIVagm-X4)] vectors were shown to specifically transduce CD4/CXCR4-positive cell lines, demonstrating the equivalent function in cell entry and choice of coreceptor usage of the V3-loops of SIVagm and HIV-1. These modified vectors were able to transduce primary human lymphocytes and were resistant to neutralization by sera from HIV-1-infected individuals. The [MLV(SIVagm-X4)] pseudotype vector generated is thus a promising candidate vector, e.g., for in vivo gene therapy of HIV-1 infection.