LPS is radioprotective in the mouse small intestine through a mechanism that includes the synthesis of cyclooxygenase-2 (COX-2) and PGE2. The goal of this study was to identify the intermediate steps in this process. We used wild-type (WT) C57BL/6 mice and knockouts for tumor necrosis factor receptors 1 and 2 (TNFR1-/-, TNFR2-/-) and recombination-activating gene 1-/- mice. Mice were given parenteral LPS and then subjected to 12 Gy total body gamma irradiation. The number of surviving intestinal crypts was assessed 3.5 days after irradiation using a clonogenic assay. Crypt cell apoptosis was assessed by histology. Parenteral administration of LPS induced COX-2 expression, PGE2 production, and radioprotection in WT and TNFR2-/- mice but not in TNFR1 -/- mice. TNFR1-/- mice were radioprotected by administration of exogenous 16,16-dimethyl PGE2. Immunohistochemical studies localized TNFR1 and COX-2 expression to subeptihelial fibroblasts and villus epithelial cells. Radiation-induced apoptosis was reduced by pretreatment with LPS in WT and TNFR2-/- mice but not in TNFR1 -/- mice. In the absence of LPS, crypt survival was elevated in TNFR1-/- when compared with WT mice. These findings demonstrate that TNFR1 function is required for LPS-induced radioprotection in C57BL/6 mice and define an essential role for TNFR1 function in the induction of COX-2 expression and PGE2 production in this process. The immunolocalization of TNFR1 and COX-2 expression to subepithelial fibroblasts following LPS administration suggests that this cell type plays an intermediate role in LPS-induced radioprotection in the intestine.