We investigated the antigen molecules for six clinically typical cases of paraneoplastic pemphigus (PNP) using immunofluorescence, immunoprecipitation, and immunoblotting. All the PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder and immunoprecipitated the 250-kD, 230-kD, 210-kD, 190-kD, and 170-kD proteins in various combinations, confirming the diagnosis of PNP. Immunoblot analysis demonstrated slightly different reactivity from that of immunoprecipitation. With immunoblotting of normal human epidermal extract, bovine desmosome preparation, and extract of cultured squamous cell carcinoma cells, all the PNP sera reacted with a characteristic doublet of the 210-kD and 190-kD proteins. However, immunoblotting detected the 250-kD desmoplakin I and the 230-kD bullous pemphigoid antigen less frequently and did not detect the 170-kD protein. Further immunoblot studies indicated that the 210-kD protein is different from desmoplakin II and that the 190-kD protein is most frequently detected by PNP sera. Two of the six PNP sera specifically reacted with the extracellular domain of recombinant pemphigus vulgaris antigen protein, indicating that pemphigus vulgaris antigen may be involved in PNP. In future studies to unravel the complex mechanisms of the PNP antigens, the immunoblot technique may be a useful tool.