Anti-p200 pemphigoid is a subepidermal immunobullous disorder associated with tissue-bound and circulating autoantibodies reactive with a 200 kDa protein on the dermal side of salt-split-skin. The autoantigen, named p200, is a non-collagenous glycoprotein located at the lamina lucida-lamina densa border of the epidermal basement membrane. However, its identity and cellular origin remain elusive. Here, we used biochemical and genetic approaches to characterize the autoantibody reactivity in three new patients with anti-p200 pemphigoid. We show that the target antigen p200 is synthesized by both keratinocytes and fibroblasts, is disulfide-bonded, and participates in calcium-dependent molecular interactions. Lack of collagen XVII (BP 180), collagen VII, or laminin 332 (laminin 5) from the dermal-epidermal junction does not destabilize p200. Colocalization within the basement membrane zone and an identical molecular weight suggested nidogen-2 as candidate autoantigen in anti-p200 pemphigoid, but biochemical analysis demonstrated that p200 is distinct from nidogen-2. In conclusion, the results define further the biochemical characteristics of p200 and demonstrate its in vitro-synthesis by keratinocytes and fibroblasts, thus providing a basis for identification and further characterization of this autoantigen.