Dialyzable leukocyte extracts (DLE) transfer specific cell-mediated immune responses from sensitized donors to non-immune recipients. In addition, DLE have several immunomodulatory effects and are used for the treatment of several infectious and non-infectious diseases. Previous studies showed that human DLE obtained from virus-infected leukocytes and bovine DLE decrease the production of the pro-inflammatory cytokine TNF-alpha in response to bacterial lipopolysaccharide, in vitro and in vivo. In the present work, we inquire as to whether DLE from uninfected human leukocytes have the ability to regulate cytokine production in peripheral blood mononuclear cells (PBMC) in vitro. We observed that PBMC from healthy individuals were able to produce TNF-alpha, IL-12 and IL-10 after stimulation with DLE. Moreover, we identified monocytes as the main cell population that produced TNF-alpha after DLE stimulation. Interestingly, we found that DLE contain unidentified ligands that activate Toll-like receptor (TLR)-2. Finally, we observed that DLE directly activated monocytes through TLR-2. These results reveal a new biological activity of DLE, and suggest that part of the immunomodulatory properties of DLE could be attributed to TLR-2 activation on monocytes and to the induction of a pro-inflammatory environment that is crucial for control of infectious diseases.