In previous studies we reported that a single injection of complete Freund's adjuvant (CFA) in early life inhibits the development of autoimmune diabetes in BB diabetes-prone (DP) rats, and that CFA upregulates tumor necrosis factor-alpha (TNFα) production by macrophages of DP rats. In this study, we asked if CFA-induced prevention of diabetes in DP rats might be mediated by TNFα. Chronic intraperitoneal injection of TNFα, 20μg daily from age 50 to 110 days, significantly decreased diabetes incidence in DP rats from 83% (15 of 18) to 37% (seven of 19) at age 110 days. Similarly, a single intraperitoneal injection of CFA, 100μl at age 26 days, significantly decreased diabetes incidence from 80% (16 of 20) to 50% (10 of 20) at age 110 days. Prevention by either TNFα or CFA was associated with decreased insulitis and preservation of islet β-cells. By contrast, treatment of DP rats with CFA, plus antiserum to TNFα three times a week from age 26 to 110 days, obviated CFA-induced protection against β-cell destructive insulitis and diabetes. Thus, diabetes incidence was 75% (18 of 24) in DP rats treated with CFA plus anti-TNFα antiserum, similar to that in untreated DP rats (80%), but only 44% (eight of 18) in DP rats treated with CFA plus control serum, similar to that in DP rats with CFA alone (50%). These results suggest that TNFα may be a mediator of the protective effects of CFA against autoimmune diabetes development in BB rats.