Eosinophil phenotypes were investigated in peripheral blood and skin lesions from eight patients with bullous pemphigoid (BP). By Nycodenz density gradients fractionation, blood eosinophils were divided into two phenotypes; normodense (greater than 1.080 g/ml) and hypodense (less than or equal to 1.080 g/ml). Increased numbers of hypodense eosinophils were observed in the blood from all patients with BP. Immunocytochemical observations, using an EG2 monoclonal antibody to react with the secretion form of eosinophil cationic protein (ECP), revealed that EG2 was expressed in 86 +/- 3% of hypodense phenotypes and 3 +/- 2% of normodense phenotypes. Ultrastructurally, hypodense eosinophils were characterized by numerous spheroidal granules, each with a lytic crystalloid core. These indicate that the hypodense phenotype represents a cell in an activated state. Only eosinophils with immunocytochemical and morphological characteristics similar to hypodense phenotypes infiltrated around the basement membrane zone in involved skin of BP. Furthermore, direct adherence of eosinophils associated with degranulation into basal keratinocytes was seen at the sites of blistering lesions. Bullous fluids contained higher concentrations of ECP than sera as determined by a radioimmunosorbent assay; thus hypodense (activated) eosinophils may directly damage the basal keratinocytes by releasing their granule proteins, subsequently leading to dermo-epidermal separation.