Previously we reported that Nef protein from human immunodeficiency virus (HIV) induces proliferation of human peripheral blood mononuclear cells (PBMC). Herein, we show that HIV replicates in cells activated by Nef. PBMC proliferated in response to Nef and were capable of producing significant amounts of infectious virus upon infection. Polyclonal antibodies to Nef synthetic peptides blocked proliferation and neutralized infection. Characteristic of superantigens, T-cell proliferation in response to Nef required antigen-presenting cells and showed CD4 Vb preferences as previously shown. These findings suggest that Nef is a virally encoded T-cell superantigen and, as such, may be vital in the establishment of HIV infection in a new host and in subsequent disease pathogenesis.