In early studies, the bullous pemphigoid antigen (BPA) has been localized extracellularly in the lamina lucida in the basement membrane zone. However, trypsin-dissociated basal cells can be tagged with bullous pemphigoid sera (BPS). By immunofluorescence, BPA appears located at the dermal pole of basal cells (BC). This may indicate that when BC are separated from the underlying matrix molecules, chunks of BPA remain attached to them. In the present study, fresh crude initial suspensions (CIS) of epidermal cells were prepared by trypsin-EDTA dissociation. The cells were smeared and air-dried. Polar fluorescent cells (i.e., BC) amounted to 42% +/- 7%. CIS were then passed through a fluorescence-activated cell sorter (FACS). In the fluorescent-positive fractions selected by FACS, 34% +/- 7% only of the BC were present. FACS-negative cell fractions were smeared on glass slides, air-dried, and restained with BPS + fluorescein isothiocyanate; 66% +/- 10% of BC were present in these fractions. This is evidence that trypsin-isolated BC comprise two subpopulations: one with BPA directly accessible, the other not. Viability tests and tissue culture studies indicated that the FACS-positive cell fractions were not viable. BPA was extracted from CIS, FACS-positive, and FACS-negative fractions and immunoblotted against BPS. Identical blots were found. FACS-negative cell fractions were treated with heparitinase, nitrous acid, methanol-chloroform, or EDTA without modifying the number of reacting cells. When BC were treated with Triton X-100 or permeabilized by successive freezings and thawings, the number of positive cells became comparable to those obtained by air-drying smears. Finally, BPA was localized on the intracellular part of hemidesmosomes of BC by immunoelectron microscopy. To see whether BPA was also present extracellularly, suction blisters were raised in minipigs and BPS injected into the blister cavity. BPA was found attached to all cells of the cellular roof but not to the dermal base of the blisters. When pieces of skin kept overnight in cold trypsin were reacted with BPS, BPA was found on both sides (epidermal and dermal) of the split. It is concluded that BPA has two localizations: one extracellular, essentially labile which accumulates at the dermal-epidermal junction; the other essentially stable which remains on the intracellular part of basal cell hemidesmosomes and which can be detected after permeabilization of the cells.