Unlike oncoretroviruses, lentiviral vectors can insert large genes and can target both dividing and nondividing cells; thus they hold unique promise as gene transfer agents. To enhance target range, the native lentiviral envelope glycoprotein is replaced (pseudotyped) with vesicular stomatitis virus G (VSVG), and the genes of interest are packaged in nonreplicating vectors by transient transfection with three plasmids. However, because of cytotoxic effects of VSVG expression in producer cells (293T cells) it has been difficult to generate a packaging cell line, required for even modest scale-up of vector production. Here we introduce a pseudotyped lentivirus vector using the baculovirus GP64 envelope glycoprotein. Compared with VSVG, GP64 vectors exhibited a similar broad tropism and similar native titers. GP64-pseudotyped vectors were usually highly concentrated without much loss of titer. Because, unlike VSVG, GP64 expression does not kill cells, we generated 293T-based cell lines constitutively expressing GP64. Our results demonstrate that the baculovirus GP64 protein is an attractive alternative to VSVG for viral vectors used in the large-scale production of high-titer virus required for clinical and commercial applications.