In systemically infected tissues of Nicotiana benthamiana, alfalfa mosaic virus (AMV) coat protein (CP) and movement protein (MP) are detected in plasmodesmata in a layer of three to four cells at the progressing front of infection. Besides the presence of these viral proteins, the plasmodesmata are structurally modified in that the desmotubule is absent and the diameter has increased drastically (almost twofold) when compared to plasmodesmata in uninfected cells or cells in which AMV infection had been fully established. Previously reported observations on virion-containing tubule formation at the surface of AMV-infected protoplasts suggest that AMV employs a tubule-guided mechanism for intercellular movement. Although CP and MP localization to plasmodesmata is consistent with such a mechanism, no tubules were found in plasmodesmata of AMV-infected tissues. The significance of these observations is discussed.