The authors have previously demonstrated spontaneous bacterial translocation (BT) in newborn rabbits and its resolution with aging. It is hypothesized that this spontaneous BT was associated with an immature gut-associated lymphoid tissue (GALT). The aim of the present study was to characterize the cellular populations of the GALT in rabbits at various ages and to correlate this with the frequency of BT. Small bowel (SB) sections and mesenteric lymph nodes (MLN) were harvested and cultured (aerobically) from New Zealand White rabbits at 0, 6, 14, 28, and more than 90 days of age for determination of bacterial colonization (BC) and BT. Portions of ileum (n = 6 for each age) were simultaneously harvested for immunoperoxidase staining. Total T cells (CDS+), expressed as the number of positive cells/1000 nuclei and activated T cells (CD25+), expressed as the number of positive cells/1000 nuclei and as the ratio of CD25+/CD5+ cells, were analyzed for each tissue. Positive cells were counted in 30 villi by light microscopy. The incidence of BT rose as BC increased in the small bowel and peaked at 6 days of age; BT then decreased with age. CD5+ cells in the small bowel villi at 0 days of age were few (2.5 positive cells/1000 nuclei) and the number significantly increased with age (6 days, 6.5; 14 days, 19.0; 28 days, 31.6; adult, 136.6 positive cells/1,000 nuclei). The distribution of T cells started in the crypts, and with advancing age, cells were found all the way to the top of the villi. The number of CD25+ cells in the villi increased with age. The CD25+/CD5+ ratio in the small bowel villi peaked at 6 days of age. These results demonstrate an inverse relationship between the number of CD5+ cells in the intestinal villi and the incidence of bacterial translocation. The elevation of activated T cells (CD25+) at 6 days of age may be the result of an immunologic activation during the time of peak bacterial translocation. These data suggest that maturity of the GALT leads to a loss of spontaneous bacterial translocation in the newborn period. Modalities that supplement the GALT may help reduce bacterial translocation.