Adenoviral vectors widely used to transfer foreign genes into neuronal cells possess tropism for glial cells 1, 2 and are toxic to infected cells. Alternatively, the use of lentiviral vectors for transducing neuronal cells has been prevailing because, in addition to their limited toxicity to infected cells, lentiviral vectors facilitate long-term expression of the transgene. The glycoprotein of lentiviral vectors that is critical for viral tropism is pseudotyped with that of vesicular stomatitis viruses (VSV-G). As the receptor for VSV-G is phosphatidylserine, it is generally thought that lentiviral vectors infect most of cell types: however, we have sometimes noted drastically different results in cell types transduced by nearly identical lentiviral vectors produced by slightly modified protocols. We found that pH of the culture media at viral harvest and lot variations in fetal bovine serum (FBS) preparations added to the culture media critically influence the resultant viral tropism. Based on our observation, this protocol provides a method that allows the production of high titer lentivectors that preferentially transduce neurons. The lentiviral vectors produced using this protocol were used in previous studies 3, 4, including re-introduction of CD38 gene expression into the hypothalamic neurons of CD38 knock-out mice 5.