The use of smokeless tobacco (moist snuff) products is associated with mucosal lesions, gingival recession, attachment loss, and oral cancer. Despite numerous reports on the general toxic effects of smokeless tobacco extract, little information is available regarding the specific effects of smokeless tobacco on immune response. Inflammatory cytokines released as a result of smokeless tobacco-induced irritation may play a role in the development of oral mucosal lesions at habitual tobacco placement sites in smokeless tobacco users. Consequently, the purpose of this study was to determine whether an aqueous extract of smokeless tobacco (STE) affects the secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and the proliferation of lymphocytes. A macrophage cell line (J774-A1) was used to measure the effects of STE on tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) secretion. Mouse splenocytes were used to assess the effects of STE on lymphocyte proliferation. We found that STE at low concentrations enhanced the production of both TNF-alpha and IL-1beta. Furthermore, STE at similar concentrations enhanced mitogen-induced murine splenocyte proliferation. Overall, these data suggest that smokeless tobacco upregulated two key proinflammatory cytokines and also induces lymphocyte proliferation.