The microbicidal activity of sodium lauryl sulfate (SLS) against human immunodeficiency virus type 1 (HIV-1) was studied in cultured cells. Pretreatment of HIV-1_NL4-3 with SLS decreased, in a concentration-dependent manner, its infectivity when using 1G5 as target cells. In the absence of a viral pretreatment period or when 1G5 cells were pretreated with SLS, the surfactant-induced inactivation of viral infectivity was less pronounced, especially at concentrations between 375 and 550 µM. SLS had no effect on HIV-1 when the virus was adsorbed to 1G5 cells by a 2-h incubation period. SLS almost completely inhibited the fusion process by decreasing the attachment of HIV-1 to target cells. SLS also inhibited the infectivity of HIV-1-based luciferase reporter viruses pseudotyped with the amphotropic murine leukemia virus envelope (which enters cells in a CD4-, CCR5-, and CXCR4-independent manner), indicating that SLS may inactivate other envelope viruses. In contrast, no effect was seen with vesicular stomatitis virus envelope glycoprotein G (which enters cells through receptor-mediated endocytosis) pretreated with up to 700 µM SLS. SLS also decreased, in a dose-dependent manner, the HIV-1-dependent syncytium formation between 1G5 and J1.1 cells after a 24-h incubation. The reduction of luciferase activity was more pronounced when J1.1 cells (which express HIV-1 proteins on their surface) were pretreated with SLS rather than 1G5 cells. Taken together, our results suggest that SLS could represent a candidate of choice for use in vaginal microbicides to prevent the sexual transmission of HIV and possibly other pathogens causing sexually transmitted diseases.