Chronic intestinal inflammation induced by 2,4,6,–trinitrobenzene sulfonic acid (TNBS) is characterized by a transmural granulomatous colitis that mimics some characteristics of human Crohn's disease. Here, we show that the transcription factor NF–κB p65 was strongly activated in TNBS–induced colitis and in colitis of interleukin–10–deficient mice. Local administration of p65 antisense phosphorothioate oligonucleotides abrogated clinical and histological signs of colitis and was more effective in treating TNBS–induced colitis than single or daily administration of glucocorticoids. The data provide direct evidence for the central importance of p65 in chronic intestinal inflammation and suggest a potential therapeutic utility of p65 antisense oligonucleotides as a novel molecular approach for the treatment of patients with Crohn's disease.