Chronic stimulation of PB T cells for 72 hours with tumor necrosis factor alpha (TNF-α) or 50% autologous SF resulted in a slight increase in basal ROS production, but did not increase intracellular ROS production to levels found in SF T cells. Exposure of PB T cells to SF (but not PB) monocytes for 72 hours, however, led to a strong increase in ROS production in PB T cells, comparable with ROS levels in SF T cells. Moreover, similar Rap1 inhibition as found in SF T cells were observed in PB T cells after exposure to SF monocytes. To demonstrate that the inhibition of Rap1 is critical in the subsequent increase in ROS production, PB T cells were nucleofected with the constitutive active isoform of Rap1 (RapV12). In RapV12 nucleofected PB T cells the SF monocyte-induced ROS production was prevented. Cell–cell contact is critical, since in PB T cells separated from SF monocytes by a transwell membrane, the inhibition of Rap1 was relieved, concomitant with an absence in excess ROS production. Additionally, we found that addition of 10 μg/ml recombinant CTLA-4-Ig fusion protein also prevented oxidative stress in PB T cells exposed to SF monocytes, which suggested a central role for CD28. PB T cells were therefore stimulated with TNF-α, interferon gamma, IL-1β, or transforming growth factor beta, in the presence or absence of anti-CD28. Here we found that stimulation with anti CD28 by itself was sufficient to induce Rap1 inhibition and induce a moderate increase in ROS production. Co-incubation of PB T cells with TNF-α strongly enhanced the intracellular ROS production.