Homeostasis in the intestinal microenvironment between the immune system and luminal antigens appears disturbed in chronic enteropathies. Pro-inflammatory cytokines likely play a role in the pathogenesis of intestinal inflammation. Several inflammatory and immunoregulatory genes have associated nuclear factor-κB (NF-κB) binding sites, which allow NF-κB to regulate gene transcription. The purpose of this study was to investigate (1) the occurrence of NF-κB activation during mucosal inflammation in situ, (2) the mucosal distribution pattern of cells expressing activated NF-κB within treatment groups, and (3) the effect of specific therapy on NF-κB activation. Dogs with chronic enteropathy were studied (n=26) and compared with 13 healthy dogs. Ten dogs had food responsive disease (FRD) and 16 had inflammatory bowel disease (IBD). NF-κB activation was detected in duodenal mucosal biopsies using a mouse monoclonal antibody (MAB 3026) that selectively binds the nuclear localization sequence of activated NF-κB. To identify macrophages, biopsies were stained using the MAC 387 antibody. Macrophages in the lamina propria double-stained for MAC 387 and NF-κB were quantitated; epithelial cell expression of activated NF-κB was determined semi-quantitatively. Results showed that more macrophages positive for activated NF-κB were present in lamina propria of dogs with chronic enteropathy compared to control dogs (p<0.01). More NF-κB positive epithelial cells were observed in FRD dogs compared to IBD dogs (p<0.05). After therapy, the number of macrophages and epithelial cells staining positive for activated NF-κB decreased (p<0.01) in chronic enteropathy dogs. In conclusion, activation of NF-κB is closely associated with the pathophysiology of canine chronic enteropathy. Down-regulation follows successful therapy.