Inflammatory bowel disease is associated with immune activation in Peyer's patches and mucosal lymph nodes. The role of these organs in dextran sodium sulfate (DSS)-induced colitis was investigated. We used mice lacking Peyer's patches and/or lymph nodes because of lymphotoxin-alpha gene deficiency or treatment in utero with lymphotoxin-beta-receptor IgG and tumor necrosis factor-receptor-I (55)-IgG fusion proteins. Mice lacking Peyer's patches and lymph nodes because of lymphotoxin-alpha deficiency or in utero fusion protein treatment developed more severe colitis than control mice as indicated by more severe intestinal shrinking, longer colonic ulcers, and higher histological disease scores. Oral DSS triggered the formation of colonic submucosal lymphoid patches in these mice and caused an increase in the number of submucosal lymphoid patches in mice treated in utero with the fusion proteins. Mice lacking Peyer's patches only showed more submucosal lymphoid patches whereas intestinal length and histological disease score were similar to control mice. In conclusion, more severe DSS-induced colitis correlates with the loss of the mesenteric lymph nodes. However, neither the absence of Peyer's patches nor the presence of colonic lymphoid patches were correlated with increased disease severity.