Regulatory T-cells (T-regs) can inhibit the immune response mediated by T-cells. There is an increasing evidence that there is an increased proportion of T-regs in PBLs and tumor-infiltrating lymphocytes in several different human malignancies, although the mechanism remains unclear. In the present study, we evaluated the prevalence of CD4(+)CD25 high T-regs in tumor-draining lymph nodes in patients with gastric cancers. Regional lymph nodes in the stomach of the patients with gastric cancer (n = 44) were classified into N1 regional lymph nodes adjacent to the gastric tumor and N2 regional lymph nodes marginally distant from the tumor. The population of CD4(+)CD25 high T-cells as a percentage of total CD4(+) cells was evaluated by flow cytometric analysis with triple-color staining. Cytokine production (IL-10 and IFN-γ) was evaluated by intracellular cytokine staining and the antiproliferative function of CD4(+)CD25(+) cells positively selected by magnetic beads was measured by evaluating the proliferative activity of CD4(+)CD25(–) cells in response to anti-CD3 plus anti-CD28 in the presence of autologous CD4(+)CD25(+) cells. The percentage of CD4(+)CD25 high T-cells in N1 regional lymph nodes (3.1 ± 0.3%) was significantly higher than that of control mesenteric lymph nodes (1.2 ± 0.3%, P < 0.01). Furthermore, a more extended area (N2) of regional lymph nodes, as well as adjacent lymph nodes (N1) to the tumors, was involved in an increased prevalence of CD4(+)CD25 high T-cells according to the disease progression. The functional evaluations confirmed that CD4(+)CD25 high T-cells derived from the lymph nodes have an inhibitory activity corresponding to T-regs. The populations of CD4(+)CD25 high T-cells in the regional lymph nodes in patients with gastric cancer were significantly higher in comparison to those in control lymph nodes. The increased prevalence of T-regs may be one of the explanations for impaired cell-mediated immunity in cancer-bearing hosts.