OX40 is a member of the TNFR superfamily (CD134; TNFRSF4) that is expressed on activated T cells and regulates T cell-mediated immune responses. In this study, we have examined the regulation of OX40 gene expression in T cells. Low-level OX40 mRNA expression was detected in both resting T cells and the nonactivated EL4 T cell line, and was up-regulated in both types of T cells upon activation with anti-CD3 Ab. We have shown in this study that basal OX40 promoter activity is regulated by constitutively expressed Sp1/Sp3 and YY1 transcription factors. NF-kappaB (p50 and p65) also binds to the OX40 promoter region, but the level of direct enhancement of the OX40 promoter activity by this transcription factor is not sufficient to account for the observed up-regulation of OX40 mRNA expression associated with activation. We have detected by chromatin immunoprecipitation that histone H4 molecules in the OX40 promoter region are highly acetylated by activation and NF-kappaB binds to the OX40 promoter in vivo. These findings suggest that OX40 gene expression is regulated by chromatin remodeling, and that NF-kappaB might be involved in initiation of chromatin remodeling in the OX40 promoter region in activated T cells. CD4(+)CD25(+) regulatory T (Treg) cells also express OX40 at high levels, and signaling through this receptor can neutralize suppressive activity of this Treg cell. In CD4(+)CD25(+) Treg cells, histone H4 molecules in the OX40 promoter region are also highly acetylated, even in the absence of in vitro activation.