Background: Factors determining the extension and degree of inflammation in the colonic mucosa of patients with ulcerative colitis (UC) are largely unknown, but CD4+CD25high regulatory T cells (Tregs) have been implicated to play an important role in suppressing inflammation. Therefore, the aims of this study were to determine whether colonic Tregs have suppressive effects on colonic effector T cells in UC and to analyze the association between segmental colonic Treg distribution and disease activity. Materials and Methods: The suppressive activity of colonic CD4+CD25high Tregs from patients with active UC was determined in coculture assays measuring proliferation and cytokine production. The frequency of Tregs and the expression of the Treg marker FOXP3 were analyzed with flow cytometry and RT-PCR in isolated cells and the whole mucosa from patients with active and inactive disease, as well as healthy mucosa. Results: Colonic CD4+CD25high T cells from patients with UC suppressed the proliferation and cytokine secretion of colonic effector CD4+ T cells. Healthy controls but not patients with UC had lower Treg frequencies in the sigmoid than in the ascending colon. Patients with UC with active disease had increased frequency of colonic Tregs. The frequency of Tregs was positively correlated with colonic disease activity and serum C-reactive protein. Conclusions: Colonic CD4+CD25high Tregs are able to suppress colonic effector T cell activity in vitro, and the Treg frequency in the inflamed intestine increases with disease activity in patients with active UC. This suggests that Tregs may be outnumbered by other inflammatory cells or that their suppressive activity may be influenced by the in vivo environment.