Activation of the lymphotoxin beta-receptor (LTbetaR), a member of the tumor necrosis factor receptor family, plays a crucial role in lymphoid organogenesis and tumor development. Lymphotoxin alpha(1)beta(2) (LTalpha(1)beta(2)) and LIGHT have been identified as membrane anchored ligands for the LTbetaR. While LTbetaR is expressed on a wide range of cell types e.g. fibroblasts and monocytes, the ligands are expressed only on activated lymphocytes and NK cells. In order to characterize LTbetaR expression and the biological consequences of LTbetaR activation rat anti-mouse LTbetaR monoclonal antibodies were generated. These antibodies recognized a mouse LTbetaR-Ig fusion protein as well as endogenous LTbetaR on a variety of mouse fibroblast and fibrosarcoma cell lines. Specificity was demonstrated by the lack of binding to LTbetaR-deficient embryonic fibroblasts. Competitive binding studies revealed that three different epitopes were recognized by the monoclonal antibodies. Two of the monoclonals activated the LTbetaR and induced activation of NFkappaB and secretion of MIP-2 and IL-6 in L929 mouse fibroblast cells. MIP-2 and IL-6 secretion was NFkappaB-dependent because IkappaB-transfected cells released significantly reduced amounts of both mediators.