Immunity against the carbohydrate components of microorganisms mediated by antibodies is an important part of host defenses. Humans and closely related primates, but not other mammals, possess natural anti-Gal1-3Gal antibodies which also, although less avidly, react with melibiose (Gal1-6Glc). Using an enzyme-linked immunosorbent assay (ELISA) with melibiose-bovine serum albumin as an antigen, we analyzed bovine anti-alpha galactosyl antibodies with respect to specificity and distribution in individual animals. Inhibition assays showed that melibiose was the strongest inhibitor, followed equally by stachyose (Gal1-6Gal1-6Glc1-2Fru) and raffinose (Gal1-6Glc1-2Fru) and then by Gal1-6Gal, Gal, and Gal1-2Gal. Others, including Gal1-3Gal and Gal1-4Gal, only exhibited minor inhibition. Thus, these bovine anti-alpha galactosyl antibodies appeared to preferentially react with Gal1-6Glc or Gal1-6Gal. The distinction of this specificity from that (Gal1-3Gal) of human antibodies was further demonstrated by the poor reaction of bovine serum to the Gal1-3Gal antigen in comparison to human serum. All 27 healthy bovine serum samples of the three age groups (newborn, calf, and adult) tested contained such antibodies with titers increasing with age. The antibodies purified by affinity chromatography using a melibiose-agarose column were mainly of the immunoglobulin G (IgG) isotype with a concentration of >23 µg/ml in most samples. IgG1 was found to be the primary antimelibiose IgG isotype in all age groups by isotype-specific ELISA, but a significant increase in IgG2, an isotype more related to innate immunity, was observed in calves and adults, compared to newborns. The purified antibodies reacted with the type II bovine strain of Streptococcus agalactiae, a common pathogen of bovine mastitis. Thus, these anti-Gal1-6Glc or Gal1-6Gal antibodies in cattle might be involved in defense against microbes bearing this or the related epitopes.