A novel canine epidermal keratinocyte cell line, MSCEK, was developed from skin of a healthy dog. The aim of this study was to determine its expression of desmosomal components and to evaluate its use as a detection tool for circulating autoantibodies in canine pemphigus. Immunofluorescence and western blotting analyses revealed that MSCEK expresses desmoglein (Dsg) 1, Dsg2, Dsg3, desmoplakin, plakoglobin and cytokeratins. Moreover, positive fluorescent reactions on the surface of MSCEK cells were observed when the cells were incubated with sera obtained from four dogs diagnosed with pemphigus complex. These findings indicate that MSCEK should be a useful tool for future research to characterize circulating autoantibodies that recognize desmosomal components in dogs with pemphigus.