Background. The successful clinical application of pig-to-primate xenotransplantation is currently limited by the development of an acute vascular rejection, which is thought to involve an induced humoral immune response to the galactose α1,3 galactose (α-Gal) antigen. Successful xenotransplantation may require the development of novel methods for removal or neutralization of anti-Gal antibodies and anti-Gal-producing B cells. The large diversity of the B-cell repertoire makes it difficult, however, to isolate and study anti-Gal B-cell development. Methods. We have established a transgenic mouse model for investigating anti-Gal B cells by introducing a transgene encoding both heavy and light chains for an anti-Gal IgM antibody into an α-galactosyltransferase-deficient (Gal-/-) background. We have characterized the frequency, phenotype, and function of transgenic anti-Gal B cells by multiparameter flow cytometric analysis and ELISA. Results. ELISA analysis of serum from animals with the transgene in an α-galactosyltransferase-deficient background (Tg Gal-/-), from transgenic animals with a heterozygous α-galactosyltransferase background (Tg Gal-/+), and from nontransgenic α-galactosyl-transferase-deficient littermates (Gal-/-) demonstrated elevated expression of anti-Gal antibodies in Tg Gal-/-mice compared with nontransgenic Gal-/-animals and a lack of transgene expression in the Tg Gal-/+mice. Anti-Gal antibody expression in Tg Gal-/-mice could be increased by immunization with an ovalbumin-Gal glycoconjugate in vivo and through stimulation with lipopolysaccharide in vitro. Multiparameter flow cytometric analysis indicates that 50% to 80% of splenic and peritoneal B cells expressed the transgene and excluded endogenous immunoglobulin gene rearrangements. The majority of these B cells expressed anti-Gal receptors on the surface, as identified by staining with a fluorescein isothiocyanate-bovine serum albumin-Gal glycoconjugate. FACS analysis of the Tg Gal-/-B cells identified them as a population of CD21highCD23lowIgMhighmarginal zone B cells in the spleen and CD5-CD23lowB1 cells in the peritoneal cavity. Conclusions. These observations suggest that this model can be used to study the regulation of anti-Gal B cells and can establish a reliable source of functional anti-Gal B cells, which could be used to test the effectiveness of α-Gal-specific immunosuppressive reagents.