To elucidate the roles of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) in oral tolerance, we studied the consequences of CTLA-4 blockade during the inductive phase of oral tolerance using a transgenic T-cell transfer model. We found that CTLA-4 blockade significantly accelerated cell cycle progression of antigen-specific T cells and dramatically increased their numbers in lymphoid organs following oral administration of ovalbumin (OVA). In mice fed with OVA, only approximately 35% of specific T cells underwent more than four cycles of cell division. This was increased to 65% in mice fed with OVA and treated with a blocking anti-CTLA-4 monoclonal antibody (mAb). The OVA-specific T cells in the latter group were localized primarily in the T-cell zones of the mesenteric lymph nodes and Peyer's patches with a few penetrated into B-cell follicles. Nevertheless, both faecal anti-OVA immunoglobulin A (IgA) and seral anti-OVA immunoglobulin G (IgG) were produced in anti-CTLA-4 mAb-treated mice. These results suggest that CTLA-4 limits the degree of T-cell activation by blocking cell cycle progression during the inductive phase of oral tolerance. In the absence of the CTLA-4 signal, mucosal exposure of antigen induces heightened T-cell activation and expansion, which in turn promotes the production of antigen-specific antibodies.