Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell-cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell-cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell-cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.