Interaction between OX40 expressed on activated T cells and its ligand (OX40L) on antigen presenting cells (APC) provides a co-stimulatory signal for T cells to promote acquired immunity. In the present study, we have examined various culture conditions for optimum OX40L expression on T cells stimulated with immobilized anti-CD3/CD28 monoclonal antibodies (mAbs). Although the day 3 primed T cells expressed minimal OX40L, after repeated stimulations both the CD4+ and CD8+ T cells became OX40L positive as determined by flow cytometry. Interleukin (IL)-12 interfered with the OX40L expression. Among activated T cells, a higher frequency of CD8+ T cells expressed OX40L than CD4+ T cells. By blocking OX40L-OX40 interaction by an anti-OX40 mAb, the number of OX40L+ T cells significantly increased. Screening of various cytokines showed that transforming growth factor (TGF)-beta1 was capable of induction of OX40L on the activated T cells within 3 days. The OX40L expressed on T cells was functional, as they bound soluble OX40 and stimulated human immunodeficiency virus-1 (HIV-1) production from cell lines chronically infected with HIV-1 and expressing OX40. Altogether the present study findings indicate that functional OX40L is inducible on human activated CD4+ and CD8+ T cells, and that the expression is enhanced by TGF-beta1.