Lentiviral (LV) vectors derived from human immunodeficiency virus type 1 (HIV-1) mediate efficient gene transfer into various cell types, including neuronal, glial, and neural stem cells in the central nervous system. The HIV-1-based LV vectors commonly used are self-inactivating and pseudotyped with a variety of viral envelope glycoproteins. Pseudotyping with vesicular stomatitis virus glycoprotein (VSV-G) confers broad tropism to wide variety of cells and enhances vector stability of the vectors. Pseudotyping with other glycoproteins alters the tropism. In particular, the use of rabies virus glycoprotein (RV-G) or fusion glycoproteins (FuGs) consisting of VSV-G and RV-G segments increases the efficiency of retrograde gene delivery into neuronal cells. The LV vector strategy provides useful approaches to genetic manipulation of specific neural pathways for elucidating the structure and function of neural circuits, and also to genome-wide screening of key molecules and gene regulatory elements in development and survival of the nervous system. Such a strategy also provides a powerful tool for clinical applications aimed at gene therapy for neurological and neurodegenerative diseases as well as central nervous system tumors.