Publisher Summary This chapter discusses the characterization of a cellular protein that promotes SV40 infection in human cells. Studies show that a cellular protein promotes SV40 infection of cultured primate cells. The protein is released into the medium by uninfected cells that are susceptible (permissive) to productive SV40 infection. This protein, designated as enhancer, facilitates the infection of human cells that exhibit low susceptibility to SV40. Addition of the SV40 enhancer before or with virus to cultured semipermissive host cells results in approximately 100-fold increases in the number of infected cells as determined by the plaquing efficiency of infectious centers and/or by immunofluorescence of nuclear SV40 T-antigen utilizing specific, fluorescein-conjugated antisera. A direct relationship exists between the susceptibility of SV40 infection and presence of active enhancer in uninfected cell cultures. Simian cells (CV-1) are very susceptible to SV40 and release easily detectable amounts of SV40 enhancer activity (10 unit/ml). In contrast, semipermissive human fibroblasts release minimal amounts of enhancer activity, and nonpermissive WISH and chick embryo fibroblast cells do not release any detectable SV40 enhancer activity. Thus, the amount of SV40 enhancer activity recovered from cell cultures coincided with the ability of SV40 to infect these cells.