The hemidesmosome is the major attachment structure of the epidermal basal cell visible ultrastructurally in skin. The importance of its components to cultured cell attachment to substratum is not understood, however. A component of the hemidesmosome, the 230-kDa bullous pemphigoid antigen (p230), has been shown to be present in an insoluble or particulate fraction of cultured cells. In order to more fully characterize its potential importance for cell-matrix adhesion in cultured keratinocytes, specific antibodies were raised to the C-terminal region of p230 expressed as a bacterial fusion protein. Such antibodies recognize the hemidesmosome of epidermis, binding on the cytoplasmic region of its plaque. In addition, keratinocytes cultured in a 0.15 mM Ca(2+)-defined medium contain a detergent-resistant pool of p230 which appears to lie in the same focal plane as the culture substrate and has a patchy or irregular distribution by indirect immunofluorescence. Treatment of cultured cells at 4 degrees C with trypsin or pronase sufficient to release keratinocytes from the culture dish does not affect the electrophoretic migration of p230 on SDS-gels, suggesting that p230 is not exposed to the extracellular space. In cells cultured in 0.15 mM Ca2+, 230-kDa BP antigen is localized to discrete clusters resting near the basal plasma membrane of the cell by immunogold staining following brief detergent treatment and fixation. These clusters are approximately 0.1 micron in diameter, which is similar in size to the in vivo hemidesmosome. Fully formed electron dense hemidesmosomal plaques are not observed under the same culture conditions, however. It appears that these clusters are early precursors of the hemidesmosome.