The autoimmune subepidermal bullous diseases are characterized by autoantibodies to the basement membrane zone of stratified squamous epithelium. Recent studies have shown that the antibodies have characteristic ultrastructural and antigenic binding properties and that differentiating between those properties can be useful in distinguishing one disease from another. Immunofluorescence microscopy is widely used to detect basement membrane zone autoantibodies. The test has traditionally used tissue substrates with an intact basement membrane zone. Those substrates are limited because autoantibody binding cannot always be detected and because autoantibodies with different ultrastructural and antigenic binding properties cannot be distinguished from each other. Normal human skin that has been separated through the basement membrane zone (i.e., split skin) has recently been used as a substrate for detecting and characterizing basement membrane zone autoantibodies by immunofluorescence. Studies indicate that split skin is a more sensitive substrate than intact skin for detecting the antibodies and that antibodies with different ultrastructural binding sites can often be differentiated from one another on split skin. Those studies suggest split skin is the substrate of choice for the routine immunofluorescence evaluation of autoimmune subepidermal bullous diseases.