Type XVII collagen, also referred to as bullous pempliigoid antigen 2 (BPAG2) or bullous pemphigoid antigen 180 (BP180), is a transmembrane protein of the hemidesmosomal complexes of keratinocytes. Type XVII collagen has an unusual type II orientation with its N-terminus intracellularly located and with a large extracellular domain that spans lamina lucida of the dermal-epidermal junction. Type XVII collagen is an autoantigen in patients with pemphigoid diseases and its gene is mutated in patients with juitctional epidermolysis bullosa. In the present work, we generated new monoclonal antibodies (MAbs) against the intracellular domain of type XVII collagen. We further characterized reactivity and fine specifity of an MAb (clone V58) from this panel of antibodies. The epitope recognized by the mAb V58 was mapped to a stretch of type XVII collagen corresponding to residues 234-398 of its sequence. Possible applications of this new MAb include antigen mapping in patients with hereditary epidermolysis bullosa and immunoaffinity purification of cell-derived type XVII collagen.