Objectives: In order to measure the immune response evoked in breast-fed infants with cow’s milk allergy (CMA) by cow’s milk challenge through human milk, mothers were given increasing doses of cow’s milk after they had been on a cow’s milk elimination diet. Another objective was to study the secretion of β-lactoglobulin (BLG) into human milk before and during milk challenge in relation to the appearance of symptoms in infants. Study design: Seventeen asymptomatic mothers who had infants with challenge-proven CMA and 10 asymptomatic mothers who had healthy infants were recruited. Infants ranged in age from 1.8 to 9.4 months. A solid-phase enzyme-linked immunoassay (ELISPOT) was used to assess the total number of immunoglobulin-secreting and specific antibody-secreting cells. Flow cytometry was used to enumerate different lymphocyte subpopulations among peripheral blood lymphocytes primed during provocation by cow’s milk antigens. BLG levels were assessed in human milk before the challenge and 1, 2, 3, and 4 hours after the commencement of the challenge. Results: All but one of the infants with CMA showed symptoms of CMA during cow’s milk challenge through human milk. There was a significant rise in the total number of immunoglobulin-secreting cells in the IgA and IgG classes associated with a positive cow’s milk challenge response, but the proportions of peripheral blood B cells bearing CD19, CD23, CD19 and 23, CD5, or CD19 and CD5 were comparable. BLG levels were comparable in both study groups. Conclusions: Most of the infants with CMA reacted to cow’s milk challenge through human milk. Hypersensitivity reactions to food antigens through human milk may be more common than previously thought. (J Pediatr 1999;135:506-12)