The synthesis of epidermolysis buUosa acquisita (EBA) antigen in simian virus 40 (SV40)-transformed human epidermal keratinocytes was studied. Indirect immunofluorescent staining of SV40-transformed keratinocytes employing a serum sample from an EBA patient as a source of antibodies decorated EBA antigen as a perinuclear granular fluorescence. This staining pattern was similar to that of nontransformed epidermal keratinocytes grown in a low Ca 2 + medium. In contrast, stratified primary cultures of keratinocytes stained after growth in a high Ca 2+ medium showed only small amounts of the antigen localized in the substrate-attached basal cells. To demonstrate biosynthesis of the EBA antigen by SV40-transformed keratinocytes, cells were metabolically labeled with 14C-amino acids and the cell lysates were immunoprecipitated with EBA antiserum. Analysis of immunoprecipitates by sodium dodeeyisulfate-polyacrylamide gel electrophoresis and fluorography revealed that the EBA serum precipitated a protein with an apparent molecular weight of 290 kD from extracts of these cells. These results indicate that SV40 induces the synthesis of the 290 kD EBA antigen. Expression of this antigen may be a general feature of nonstratifying, proliferating epidermal cells.