T lymphocytes expressing NK1.1 marker (NK1.1+) have been suggested as being important in peripheral immune modulation. Alteration of the balance between Th1 proinflammatory and Th2 anti-inflammatory cytokine-producing cells can ameliorate immune-mediated disorders. The aim of the study was to determine the role of NK1.1+ lymphocytes in the pathogenesis of tolerance and proinflammatory states and to determine their role in altering the Th1/Th2 balance in experimental colitis. Colitis was induced in C57/B6 mice by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Mice received five oral doses of colonic proteins extracted from TNBS colitis colonic wall. Standard clinical, macroscopic, and microscopic scores were used for colitis assessment. Liver-associated lymphocytes and splenocytes were harvested 14 days following tolerance induction. Depletion of NK 1.1+ lymphocytes was performed 36 hr before lymphocyte harvesting. Lymphocytes were cultured for 12 hr with Con A and colitis extracted proteins. To evaluate the role of NK1.1+ lymphocytes in keeping a balance between immunogenic and tolerogenic subsets of cells, intracellular staining and flow cytometry assays were performed in tolerized and nontolerized mice. IL-4, IL-12, and IFN-gamma levels were measured by ELISA. Administration of mouse-derived colitis-extracted proteins ameliorated experimental colitis. Tolerized mice exhibited significant improvement in all macroscopic and microscopic parameters for colitis. Depletion of NK1.1 following tolerance induction significantly decreased the CD4(+)IL-4(+)/CD4(+)IFN-gamma(+) ratio in tolerized mice. However, depletion of NK1.1 lymphocytes in nontolerized mice increased the CD4(+)IL-4(+)/CD4(+)IFN-gamma(+) ratio, compared with nondepleted nontolerized mice. Induction of tolerance led to an increase in IL4 and a decrease in IFN-gamma levels. In the experimental colitis model NK1.1+ lymphocytes play a dual role: In the presence of peripheral tolerance they may be accountable for keeping the high CD4(+)IL-4(+)/CD4(+)IFN-gamma(+) ratio and disease alleviation. However, in nontolerized conditions they may induce a proinflammatory shift.