Flow cytofluorometric analyses of lymphocytes harvested from the mesenteric lymph node (MLN), mucosal epithelium, and lamina propria of C57BL/6 mice demonstrate that expression of alpha/beta or gamma/delta T-cell receptors (TCR) and CD4 or CD8 molecules by T lymphocytes in the intestinal immune system varies depending upon their anatomic location. The MLN contained equivalent numbers of CD4+ and CD8+ T cells, the vast majority of which were alpha/beta TCR positive (alpha/beta TCR+). The lamina propria T cells were predominantly CD4+ and alpha/beta TCR+, while the intestinal intraepithelial lymphocytes consisted of equivalent numbers of alpha/beta and gamma/delta T cells, the majority of which were CD8+. There were no significant changes in these T-cell phenotypic profiles when the mice were antibiotic decontaminated or monoassociated with Escherichia coli. Mice were depleted of CD4+ T cells and/or CD8+ T cells in vivo by intraperitoneal injections of monoclonal antibody GK 1.5 (rat anti-mouse CD4) and/or monoclonal antibody 2.43 (rat anti-mouse CD8). T-cell depletion was confirmed in the MLN, lamina propria, and the intestinal epithelium by flow cytometry. E. coli C25 translocation from the gastrointestinal (GI) tract to the MLN was significantly increased in mice depleted of CD4+ T cells, CD8+ T cells, or both. T-cell-deficient athymic beige/nude mice also exhibited greater levels of E. coli C25 translocation to the MLN than beige/het euthymic littermates. Salmonella typhimurium translocation also was increased following CD4+ and CD8+ T-cell depletion in mice monoassociated with S. typhimurium. Depletion of CD4+ and/or CD8+ T cells also increased the translocation to the MLN of certain indigenous GI flora bacteria. These results confirm that T-cell-mediated immunity is involved in the host defense against bacterial translocation from the GI tract.