The infection and subsequent replication rates of human immunodeficiency virus type 1 (HIV-1) affect the pathogenicity. The initial stage of HIV-1 infection is largely regulated by viral envelope sequence. We previously reported that the defective doughnut-shaped particles produced from a persistently infected cell clone, named L-2, obtained from human CD4+ T-cell line MT-4 that was persistently infected with HIV-1 LAI strain, efficiently form particle-mediated syncytia with uninfected human CD4+ T-cell line, MOLT-4. Here, we prepared a molecular clone (pL2) containing the L-2 provirus to characterize the viral genetic region contributing to this activity to form particle-mediated syncytia. Several recombinants were constructed with pNL4-3 by replacing the pL2-derived region including full-length env. Characterization of the particles obtained by transfection with these recombinant clones confirmed that pL2-derived env carried the particle-mediated syncytia formation activity. It is noteworthy that the pL2-derived env region could also contribute to enhancement of infectivity in CD4+ T-cell lines as well as primary peripheral blood mononuclear cells (PBMCs). Thus, the HIV-1 particle-mediated syncytium formation activity could also contribute to the enhancement of HIV-1 infectivity.