We have previously shown that CD4+ T cells are required to optimally expand viral-specific memory CD8+ CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4+ T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4+ T cells, which subsequently can help CD8+ T cell responses. The current study examined whether OX40 ligation on ex vivo CD4+ T cells can enhance their ability to “help” virus-specific CTL responses in HIV-1-infected and-uninfected individuals. OX40 ligation of CD4+ T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and-uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4+ T regulatory cells, but was associated with a direct effect on proliferation of CD4+ T cells. Thus, OX40 ligation on CD4+ T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.