Lentiviral Nef increases viral replication in vivo, plays a direct role in pathogenesis, and increases viral particle infectivity. We now find that HIV Nef also increases the activation of T cells, a cellular state required for optimal viral replication. This enhancement is stimulant-dependent. As defined by IL-2 generation, activation of T cells stimulated with classical mitogens [phorbol 12-myristate 13-acetate (PMA) + anti-CD3, PMA + phytohemagglutinin, and PMA + ionomycin] is unaffected by the expression of Nef. However, Nef increases IL-2 secretion when cells are stimulated through the T cell receptor and the costimulus receptor (CD28). This increase in activation, which depends on Nef myristylation, is caused by an increase in the number of cells reaching full activation and not by an increase in the amount of IL-2 secreted per cell. These findings demonstrate that Nef lowers the threshold of the dual-receptor T cell activation pathway. The capacity of Nef to increase T cell activity may be very important in vivo when Nef is the predominant or the only viral gene product expressed.