OX40 is a member of the tumor necrosis factor (TNF) receptor superfamily and known to be an important costimulatory molecule expressed on activated T cells. To investigate the role of costimulation of OX40 in human immunodeficiency virus type 1 (HIV-1) infection by its natural ligand, gp34, the OX40-transfected ACH-2 cell line, ACH-2/OX40, chronically infected with HIV-1, was cocultured with paraformaldehyde (PFA)-fixed gp34-transfected mouse cell line, SV-T2/gp34. The results showed that HIV-1 production was strongly induced. This was followed by apparent apoptosis, and both processes were specifically inhibited by the gp34-specific neutralizing monoclonal antibody 5A8. Endogenous TNF alpha (TNF-) and TNF- production were not involved in the enhanced HIV-1 production. Furthermore, enhanced HIV-1 transcription in gp34-stimulated ACH-2/OX40 cells was dependent on the B site of the HIV-1 long terminal repeat, and the OX40-gp34 interaction activated NF-B consisting of p50 and p65 subunits. When primary activated CD4⁺ T cells acutely infected with HIV-1_NL4-3 (CXCR4-using T-cell-line-tropic) were cocultured with PFA-fixed gp34⁺ human T-cell leukemia virus type 1-bearing MT-2 cells or SV-T2/gp34 cells, HIV-1 production was also markedly enhanced. The enhancement was again significantly inhibited by 5A8. The present study first shows that OX40-gp34 interaction stimulates HIV-1 expression and suggests that OX40 triggering by gp34 may play an important role in enhancing HIV-1 production in both acutely and latently infected CD4⁺ T cells in vivo.