Although two groups of bullous pemphigoid antigens have been well characterized, different research groups have shown strikingly different prevalence rates of antibodies to these antigens in their patients. Potential explanations for this phenomena include a patient population that has different prevalence of antibodies, or that the antigen preparations used by the different groups contain different relative amounts of these antigens. We have compared the relative concentration of the different bullous pemphigoid antigens in epidermal extract preparations made by three different procedures commonly used to separate dermis from epidermis: NaCl, ethylenediaminetetraacetic acid (EDTA), and heat. We have found that the amount of the 180-kD antigen present in extracts is dependent on the techniques involved in separation of the epidermis from dermis. NaCl- and EDTA-separation procedures result in partial proteolysis of the 180-kD antigen to smaller forms, including major brands at 160 kD and 97 kD in the EDTA preparation. Fragments of the 180-kD antigen are present in both the separation and wash fluids, associated with a significant reduction of the 180-kD form in the extract of the NaCl-separated skin. We conclude that the native molecular weight of the previously described minor bullous pemphigoid antigen is 180 kD, and that the apparent difference in patient reaction to the 180-kD antigen may be due to different preparations of the antigen rather than underlying differences in seropositivity in the patient population.