Objective: The main objective of the present study was to explore contact-independent immune suppressive mechanisms by humoral factors in malignant ascites from ovarian carcinoma that target effector T cells. Methods: Flow cytometry was the main method used to detect T cell function as assessed by CFSE-proliferation rate, in the presence of different concentrations of ascites fluid. Cell-free ascites was sometimes pretreated using a biochemical approach. Different antibody inhibitors were used to reverse the ascites-induced inhibition of T cell proliferation. Results: By culturing T cells in cell-free ascites we demonstrated malignant ascites fluid to be highly immunosuppressive both against autologous and allogeneic T cells (n=6, p<0,001). The inhibitory factor(s) in ascites did not appear to be secreted from the ovarian carcinoma (OC) cells, as transfer of culture media from tumor cells isolated from OC patients or the ovarian cancer cell-line SKOV-3 did not suppress effector T cell functions in vitro. A more detailed characterization demonstrated that the inhibition could not be reversed by targeting potential suppressive mechanisms like IL-6, IL-8, IL-10, CTLA-4, PD-1, B7-DC, B7-H1 or PI3K (n=5). Furthermore, we found the inhibitory factor(s) to be sensitive to proteases and denatured by heat and acetone (n=3). Conclusion: In conclusion, our data indicate the presence of a yet unknown inhibitory protein factor(s) in malignant cell-free ascites, not secreted by tumor cells, but possibly by immune cells such as regulatory T cells (Tregs).