Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by IgG autoantibodies against desmoglein 3 (Dsg3). In this study, we characterized the ultrastructural localization of in vivo-bound IgG, Dsg3, and desmoplakin during the process of acantholysis in an active mouse PV model, using post-embedding immunoelectron microscopy. In non-acantholytic areas of keratinocyte contact, IgG labeling was restricted to the extracellular part of desmosomes, and was evenly distributed throughout the entire length of the desmosome. The distribution of in vivo IgG was similar to that of anti-Dsg3 labeling in the control mouse. Within the acantholytic areas, there were abundant split-desmosomes with keratin filaments inserted into the desmosomal attachment plaques. These split-desmosome extracellular regions were also decorated with anti-Dsg3 IgG and were associated with desmoplakin staining in their cytoplasmic attachment plaques. No apparent split-desmosomes, free of IgG-labeling were observed, suggesting that Dsg3 was not depleted from the desmosome before the start of acantholysis in vivo. Desmosome-like structures (without keratin insertion) were found only on the lateral surfaces of basal cells, but not on the apical surfaces at the site of acantholytic splits. These findings indicate that anti-Dsg3 IgG antibodies can directly access Dsg3 present in desmosomes in vivo and cause the subsequent desmosome separation that leads to blister formation in PV.