We have previously reported that a single injection of complete Freund's adjuvant (CFA) can prevent diabetes appearance in diabetes-prone (DP) BB rats. In this study, we investigated further the mechanism of CFA-induced protection from diabetes. We found that adoptive transfer of splenic cells from CFA-treated DP rats into young DP rats protected the latter from diabetes development. This suggested that CFA-induced protection from diabetes resulted from activation of regulatory (suppressor) cells. Cell mixing experimentsin vitroindicated that CFA activated splenic cells with antigen-nonspecific suppressor activity (suppression of lymphoproliferative responses to lipopolysaccharide and to allogeneic splenic cells). Fractionation of splenic cells on Percoll revealed that the suppressor activity resided in low density cells relatively depleted of T-cells, B-cells, macrophages and NK cells. These results suggest that non-specific (natural) suppressor cells in CFA-treated BB rats may be responsible for suppressing autoimmune responses and preventing insulitis and diabetes development.