T-lymphocytes are considered to play an essential role in the pathogenesis of inflammatory bowel disease. The goal of this study was an objective determination of the distribution of gastrointestinal CD3+ lymphocytes in a standardized dog population as a gold standard for further investigations. Full thickness biopsies were obtained from seven different localizations from stomach to colon from six adults and four neonatal healthy Beagle dogs. The tissue was stained with an anti-CD3 antibody. The positive cells were counted in an area of 200,000 μm2 at four different sites per localization. In adult dogs, the horizontal distribution showed a maximum of CD3+ lymphocytes in the duodenum and jejunum. In the stomach, almost no positive cells were observed. The vertical distribution revealed an accumulation in the villi. The neonatal dogs showed a similar distribution pattern, but on average ten times less CD3+ lymphocytes in the analogous localizations and smaller differences between localizations.