A mutant of the Cucumber mosaic virus subgroup IA strain Fny (Fny-CMV) lacking the gene encoding the 2b protein (Fny-CMVΔ2b) induced a symptomless systemic infection in tobacco. Both the accumulation of Fny-CMVΔ2b in inoculated tissue and the systemic movement of the virus appeared to proceed more slowly than for wild-type Fny-CMV. The influence of the 2b protein on virus movement in the inoculated leaf was examined using viral constructs derived from Fny-CMV and Fny-CMVΔ2b expressing the green fluorescent protein. Laser scanning confocal microscopy was used to visualize the movement of these viruses. Whereas the wild-type virus spread between the epidermal cells as well as the mesophyll cells, the mutant virus spread less efficiently through the epidermal layer and moved preferentially through the mesophyll. Thus, the 2b protein of Fny-CMV influences the dynamics of movement of the virus both within the inoculated leaf and through the whole plant. We propose that this altered movement profile of Fny-CMVΔ2b results in the absence of disease symptoms in tobacco.