Background: The role of exposure to dietary antigens through maternal milk is intriguing, because it may result either in immunization or in tolerance. Exposure to cow's milk proteins results in antibody formation against bovine insulin in infants at risk for type 1 diabetes. Objective: To study the appearance of IgG antibodies to bovine and human insulin in infants with an atopic family history whose mothers followed a cow's milk–free diet during the first 3 months of lactation. Methods: In a prospective cohort study on prevention of food allergies, 123 infants were exclusively breast-fed or received supplementation with a hydrolyzed casein-based formula (Nutramigen) until the age of 6 months. The mothers either avoided cow's milk during the first 3 months of lactation (diet group) or had an unrestricted diet (nondiet group). The levels of IgG antibodies to bovine and human insulin were determined by enzyme immunoassay at 3, 6, 12, and 18 months and at 4 years. In addition, cord blood was obtained at birth and a maternal sample at delivery. Results: At 3 months, IgG antibodies to bovine insulin were low in both dietary groups (median levels 0.150 and 0.114 optical density units in the diet and nondiet groups). After exposure to dietary insulin, IgG antibodies to bovine insulin increased in both groups, reaching a peak at 12 months in the nondiet group and at 18 months in the diet group. At 18 months, IgG antibodies to bovine insulin were lower in infants in the nondiet group than in infants in the diet group (0.287 vs 0.500, P < .0001). At 4 years, the antibodies no longer differed between the groups. Conclusion: The exposure to cow's milk proteins through breast milk during the first 3 months of life resulted in decreased levels of antibodies to dietary bovine insulin at 18 months of age, suggesting a role for breast milk antigens in early tolerance induction. (J Allergy Clin Immunol 2000;106:302-6.)