Previous studies have suggested that oral tolerance induction by low doses of Ag is mediated by inhibitory cytokines, particularly TGF-β1. To examine the roles of TGF-β1 and other inhibitory cytokines in the induction of oral tolerance, TGF-β1 null mice and controls were gavaged with 10 to 20 mg (high dose) or 1 mg (low dose) of OVA for 3 days. After immunization with OVA, the in vitro proliferative response of OVA-specific popliteal lymph node cells was assessed. Lymphocytes from all TGF-β1 null mice fed high doses of OVA exhibited highly significant suppression compared with controls. A weaker, but still significant, suppression was observed in lymphocytes from the majority of TGF-β1 null mice fed low doses of OVA. In addition, supernatants from these lymphocytes exhibited lower levels of IL-4, IL-10, and IFN-γ than those from water-fed control animals. These results indicate that while TGF-β1 may play a role in suppression, inhibitory cytokines are not the exclusive mechanism by which low dose oral tolerance is induced.