Extracorporeal photopheresis (ECP) is an immunomodulatory cell therapy being investigated as a treatment for various immune-mediated inflammatory disorders. Clinically, ECP involves the intravenous reinfusion of autologous, apoptotic peripheral blood leukocytes. The exact mechanism of action behind ECP, however, remains unclear. In animal models, delivery of apoptotic cells has been shown to regulate immune responses through the downregulation of antigen-presenting cell (APC) function, the modulation of cytokines, and the generation of regulatory T cells. We and others, have shown that activation of naive T cells in the presence of APCs that have engulfed ECP-treated apoptotic cells leads to the generation of a T cell population that can suppress syngeneic T cell proliferation and function. In the present study, we demonstrate that the direct interaction of ECP-treated peripheral blood mononuclear cells (PBMCs) with naive human CD4+ T cells in vitro also promotes a T cell phenotype with regulatory activity. Transfer of ECP-derived regulatory T cells to a secondary mixed lymphocyte reaction results in a greater than 50% inhibition of syngeneic responder T cell proliferation and IFNg production at a Treg:responder ratio of 1:10. In addition, transwell assays demonstrate that inhibition of responder T cells by ECP-derived regulatory T cells is contact-dependent. The generation of human regulatory T cells by ECP is dependent on the presence of ECP-treated monocytes and can be reversed by the addition of IL-2. These regulatory T cells also proliferate in response to concanavalin-A, but do not express increased levels of Foxp3 or IL-10, nor do they secrete significant levels of the pro-inflammatory cytokines IL-2, IFNg, IL-4, and TNFa. Additional studies are underway to further characterize the phenotype of these regulatory cells and to determine their suppressive capacity in vivo. It is hoped that a better understanding of the mechanism of action behind ECP will help to define more efficacious ECP treatment regimens.