The subcellular locations of two potyviral proteins, the coat (CP) and nonstructural cylindrical inclusion (CI) proteins of tobacco vein mottling virus (TVMV), during early stages in the development of systemic infections in plants, have been investigated. Ultrathin sections of newly emerged leaves in infected plants were treated with antibodies specific to these proteins and then with gold-labeled secondary antibodies and examined by electron microscopy. CI was detected near plasmodesmatal connections between mesophyll cells prior to the appearance of CP or any virus-induced features or effects. Further accumulation of CI was evident in the form of conical structures, many of which appeared to penetrate the cell wall and to be connected to cones in neighboring cells. Prior to its appearance in other parts of the cells, the viral CP was detected, often in linear arrays, near the vertices or inside the cones and in plasmodesmata. In situ hybridization analysis of similar tissue sections with a TVMV RNA-specific oligoribonucleotide probe revealed the presence of the viral RNA in plasmodesmata. These results lend support to the notion that the formation of specific structures by potyviral CI proteins is required for and plays a direct role in the intercellular passage of viral genetic material, in the form of virus particles or complexes containing viral CP and RNA, in infected plants.