The desmoglein plays an important part in the formation of desmosomes. We constructed recombinant adenoviruses containing desmoglein 1 and desmoglein 3 derivatives partly lacking the extracellular domain (desmoglein 1DeltaEC and desmoglein 3DeltaEC, respectively), and full-length desmoglein 1 and desmoglein 3 and studied the involvement of desmoglein 1 and desmoglein 3 in desmosome formation. During low-level expression of desmoglein 3DeltaEC in transduced HaCaT cells, keratin insertion at cell-cell contact sites was only partially inhibited and desmoplakin was partially stained at cell-cell contact sites. Low-level expression of desmoglein 1DeltaEC, however, resulted in complete inhibition of keratin insertion at the cell-cell contact sites, and desmoplakin was stained in perinuclear dots. These results indicate the dominant-negative effect of desmoglein 1DeltaEC on desmosome formation was stronger than that of desmoglein 3DeltaEC. Desmoglein 1DeltaEC coprecipitated plakoglobin to approximately the same extent as desmoglein 3DeltaEC. Therefore, we conclude that the dominant-negative effect of desmoglein 1DeltaEC is not simply due to plakoglobin sequestration. On the other hand, during low-level expression of full-length desmoglein 3 and desmoglein 1, they both colocalized with desmoplakin. During high-level expression, however, keratin insertion at cell-cell contact sites was inhibited in desmoglein 1 but not in desmoglein 3, and desmoplakin was stained at cell-cell contact sites in desmoglein 3 but not in desmoglein 1. These data suggest desmoglein 1 and desmoglein 3 expressed at low level were incorporated into desmosome but at high-level expression, desmoglein 1 disrupted desmosomes but desmoglein 3 did not. Our findings provide biologic evidence that desmoglein 1 and desmoglein 3 play a different functional role in cell-cell adhesion of keratinocytes.