Background: Evaluation of immune responses to food proteins in animal models requires that the animals are not already sensitized or orally tolerized against the proteins in question. Since maternal transfer of specific immune responses has been observed, breeding of animals on an antigen-free diet for several generations may be necessary to obtain immunologically naïve animals. Methods: To determine the most appropriate breeding conditions of mice to be used in immunological studies on food proteins, we examined immune responses towards β-lactoglobulin (BLG) in mice bred on a milk-containing diet (F0) and then for three generations (F1-F3) on a commercially available milk-free diet. The specific antibody and cell-proliferative response to BLG was compared in non-immunized and immunized BALB/c mice, and in mice orally tolerized to BLG prior to immunization. Results: The immune response to BLG in the F1 generation deviated from the response observed in the F0 and F2/F3 generations. Importantly, trace amounts of BLG detected in the commercial milk-free diet did not induce oral tolerance. Conclusions: The study showed that breeding mice on an antigen-free diet for at least two generations is required to attain animals appropriate for immunological studies of food proteins. Although the small quantity of BLG in the milk-free diet did not induce detectable oral tolerance in the present study, it is strongly recommended that the potential effect of contaminating dietary antigen is considered in future studies on food proteins.