question: Cholesterol, a major component of plasma membrane lipid rafts, is important for assembly and budding of enveloped viruses, including influenza and HIV-1. Cholesterol depletion impairs virus assembly and infectivity. This study examined the effects of exogenous cholesterol addition (delivered as a complex with methyl beta cyclodextrin) on the production of Molony murine leukemia virus retroviral vector and HIV-1-based lentiviral vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Cholesterol supplementation before and during vector production enhanced the infectivity of retroviral and lentiviral vectors up to 4-fold and 6-fold, respectively. In contrast, the amount of retroviral vector produced was unchanged, and that of lentiviral vector was increased less than two-fold. Both free cholesterol and cholesterol ester content in 293-gag-pol producer cells increased with cholesterol addition. In contrast, the phospholipids headgroup composition was essentially unchanged by cholesterol supplementation in 293-gag-pol packaging cells. Based on these results, it is proposed that cholesterol supplementation increases the infectivity of VSV-G-pseudotyped retroviral and lentiviral vectors, possibly by altering the composition of the producer cell membrane where the viral vectors are assembled and bud, and/or by changing the lipid composition of the viral vectors.