Several lines of evidence suggest that leukocyte adhesion molecules can promote HIV-1-mediated cell fusion and syncytium formation. In the present studies, the human kidney cell line, 293, was transfected with the envelope glycoprotein gene of the MN strain of HIV-1 alone or cotransfected with a cDNA encoding intercellular adhesion molecule 1 (ICAM-1). It was found that 293 cells transfected with the HIV-1MN env gene expressed the HIV-1 polyglycoprotein precursor, gp160, and the mature gp120-gp41 complex. When mixed with a CD4+ T cell line (CEM), the gp160-transfected cells mediated heterotypic cell fusion and formed multinucleate syncytia. Virus-neutralizing monoclonal antibodies to the V2 and V3 domains of gp120 were able to inhibit syncytium formation, as were monoclonal antibodies to CD4. When ICAM-1 was coexpressed with gp160, syncytium formation between the transfected kidney cells and uninfected CD$+ T cells was markedly enhanced. Inhibitors of HIV-1 infectivity (e.g., monoclonal antibodies to gp120, recombinant soluble CD4) were able to prevent syncytium formation; however, the syncytium-blocking activity of these agents was significantly attenuated in cultures in which ICAM-1 was cotransfected with gp160. These results confirm that leukocyte adhesion molecules can promote gp160-mediated syncytium formation and demonstrate, for the first time, that adhesive interactions mediated by ICAM-1 and its contrareceptor, LFA-1, attenuate the syncytium-inhibiting activity of virus-neutralizing monoclonal antibodies and soluble CD4. These findings suggest that the type and magnitude of leukocyte adhesion molecules expressed on cells may be a significant variable in in vitro HIV-1 neutralization assays.