Some neuronal-derived CD4-negative cells are susceptible to infection with human immunodeficiency virus type 1 (HIV-1). Galactosyl ceramide is an alternate receptor for HIV-1 that appears to bind in vitro to the C2, V3, V4, and V5 regions of gp120. Amino acid variation in the V3 loop of HIV-1 affects cellular tropism in CD4-positive cells, but its effect on CD4-negative cells has not been fully analyzed. Here, we describe the effect of amino acid changes in V3 on the HIV-1 infection of a CD4-negative neuronal cell line, SK-N-MC. The sequence of the V3 domain was found to dramatically alter virus infectivity. Furthermore, a gp120 V3 loop neutralizing monoclonal antibody blocked HIV-1 infection of SK-N-MC cells. This data suggests that V3 may also serve as a primary viral determinant for infectivity of CD4-negative cells.