Intestinal inflammation in inflammatory bowel disease (IBD) and experimental models of colitis is characterized by a dysregulated intestinal immune response with elevated levels of Th₁ cytokines. The luminal flora has been implicated as a major factor contributing to the initiation and perpetuation of inflammation in experimental colitis by mechanisms not known. Bacterial DNA contains unmethylated cytosin–guanosin dinucleotides (CpG) which strongly activate Th₁-mediated immune responses. To test whether these CpG-motifs modulate intestinal inflammation we treated mice with dextran sulphate sodium (DSS)-induced colitis with CpG-containing oligodeoxynucleotides (CpG-ODN). CpG-ODN given after the onset of DSS colitis aggravated the disease, as indicated by a significantly increased loss of body weight and a 30% increase of the histological score. Further, we found a severe increase of proinflammatory cytokines (interleukin (IL)-6: 40-fold; interferon (IFN)-γ : 11-fold). In a pretreatment setting CpG-ODN reduced weight loss significantly and reduced intestinal inflammation by 45%. Colonic IFN-γ and IL-6 mRNA levels were reduced by 75%, and IL-10 was elevated by 400% compared to controls. The prophylactic CpG-effect was not imitated by IL-12 because IL-12 pretreatment was not protective. In time-course experiments, CpG-ODN pretreatment over 5 days resulted in a tolerance effect concerning its IFN-γ-inducing quality, and during the following days of colitis induction IL-10 secretion from mesenterial lymph node cells was elevated compared to controls. Therefore, the prophylactic effect of CpG-ODN might be explained by its tolerizing effect and/or the increased ability for IL-10 production during the consecutive intestinal inflammation.