Exposure of humans to topical bovine thrombin has been associated with development of antibodies against bovine and human coagulation factors and blood coagulation abnormalities. However, the nature of this humoral response is unknown. In this study, numerous glycoproteins in the topical bovine thrombin were found to contain the Gal(alpha1)-3Gal epitope, which is known to be highly immunogenic. More importantly, Gal(alpha1)-3Gal is recognized by natural antibodies that are found in all normal individuals and are known to effectively mediate complement activation and subsequent destruction of xenogeneic tissues. Thus, primary exposure of normal individuals to topical bovine thrombin is expected to result in an immediate immune reaction against that reagent. Further, following exposure to topical bovine thrombin, the levels of anti-Gal(alpha1)-3Gal IgG rose to levels tenfold greater than the average level of natural anti-Gal(alpha1)-3Gal IgG in naive individuals. Thus, Gal(alpha1)-3Gal in topical bovine thrombin accounts for, at least in part, the highly immunogenic nature of this reagent.