The expression and subcellular location of the 33.6-kDa nonstructural protein NSm of tomato spotted wilt virus (TSWV) was analyzed in Nicotiana rustica plants and protoplasts as a function of time. Immunofluorescent studies in protoplasts isolated from TSWV-infected N. rustica leaves showed that this protein could first be detected close to the periphery of the cell, near the plasmamembrane, and later in tubular structures emerging from the cell surface. In situ, these tubules appeared specifically in the plasmodesmata, suggesting their involvement in cell-to-cell movement of the virus during systemic infection. In protoplasts transfected with an expression vector containing the NSm gene, similar tubules were formed, indicating that NSm has the ability to form these structures in the absence of other virus-specific components. To test whether plant-specific components were involved in tubule formation, the NSm gene was also expressed in a heterologous expression system, i.e., insect cells. Spodoptera frugiperda and Trichoplusia ni cells were infected with a recombinant baculovirus expressing the NSm-gene (AcNPV/NSm). The efficient formation of NSm-containing tubules emerging from the surface of both cell types indicate that no plant-specific cell structures or proteins are involved in their development.