Lentiviral vectors are increasingly used for gene delivery to neurons and in experimental models of neurodegeneration. Their use in gene delivery to white matter and their potential value in preventing or repairing CNS demyelination has received less attention. Here we show using a VSV-G-pseudotyped HIV-derived vector expressing the marker gene LacZ that lentiviral vectors transduce the major macroglial cell types present in normal white matter (astrocytes, oligodendrocytes, and oligodendrocyte progenitors). Injection of lentiviral vectors causes an inflammatory response at the injection site characterized by OX42+ and ED1+ macrophages, but only a few CD8+ and no CD4+ lymphocytes, and mild demyelination. Injection of lentiviral vectors into areas of toxin-induced demyelination resulted in significant numbers of cells expressing the marker gene and was a more effective means of gene delivery than was a LacZ-expressing murine retroviral vector.