Roles of cytokines in primary and secondary Dengue virus (DV) infections are not completely understood. In this study, we challenged mononuclear leukocytes (MNLs) obtained from non-immune and DV-1-infected donors with DV-2 in vitro to mimic primary and heterologously secondary DV-2 infections, respectively. We found that MNLs in response to DV-2 could release a large amount of interleukin-1 (IL-1 beta) and tumor necrosis factor alpha (TNF alpha). However, IL-1 beta and TNF alpha released by MNLs with primary or heterologously secondary exposure to DV-2 were not significantly different. In contrast, MNLs with heterologously secondary DV-2 infections produced significantly higher amount of interferon gamma (IFN gamma) than those with primary DV-2 infections. These results suggest that IFN gamma, but not TNF alpha or IL-1 beta, may in part participate in the pathogenesis of Dengue shock syndrome (DSS) frequently found in heterologously secondary DV-2 infections.