Oral administration of Ag results in systemic hyporesponsiveness termed oral tolerance. The regulatory cells induced by oral Ag are effective in the suppression of Th1-type autoimmune diseases. We examined the cytokine microenvironment in gut-associated lymphoid tissue in response to orally administered OVA in OVA TCR-transgenic mice. Mice were fed a low (0.5 mg) or high (500 mg) dose of OVA one time or five times. Immunohistochemical analysis demonstrated increased IL-4, IL-10, and TGF-beta in the gut within 6 h of a low-dose feeding and after five low-dose or high-dose feedings. IFN-gamma and IL-2 either decreased or showed no change, with the exception of a small transient increase in IL-2 at 6 h after a low dose. Increases in IL-4 and IL-10 were found in the dome of the Peyer's patch, and increases in TGF-beta were observed in the interfollicular region and the villi. IL-10 was also substantially increased in the villi. IL-4 and IL-10 were produced predominately by CD4+ T cells. TGF-beta was found predominately in macrophages and CD4+ T cells. Peyer's patches had a marked up-regulation of TGF-beta mRNA as measured by RT-PCR. These results demonstrate the differential activation of cytokine production in discrete regions of gut-associated lymphoid tissue. The induction of cytokines known to inhibit autoimmune disease at the site of Ag absorption indicates an important role for the mucosal immune system in the establishment of oral tolerance.