Germfree mice were fed through three generations a water soluble, chemically defined, "antigen-free" diet with a supplement of oil and oil-soluble vitamins. Second generation animals were compared to germfree and specific-pathogen-free mice fed a natural-type commercial diet. The ceca of the germfree mice fed the antigen-free diet were smaller than those of germfree mice fed the natural-type diet but larger than those of specific-pathogen-free mice fed the natural-type diet. Their mean spleen size was between that of germfree and specific-pathogen-free mice fed the natural-type diet, but the differences were of borderline significance. Germfree mice fed the antigen-free diet had fewer leukocytes than the other groups. Their serum immunoglobulin G level was one-tenth that of germfree mice fed the natural-type diet and one-hundredth that of specific-pathogen-free mice fed the natural-type diet. Their serum immunoglobulin M was only slightly below germfree, natural-type diet levels. Immunoglobulin A could be detected in the intestinal wall and contents of specific-pathogen-free mice fed the natural-type diet but not in either of the germfree groups.