Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are assembled in modified plasmodesmata. Similar tubular structures are formed on the surface of protoplasts inoculated with cowpea mosaic virus. The RNA 2-encoded movement protein (MP) is responsible for the induction and formation of these structures. To define functional domains of the MP, an alanine-substitution mutagenesis was performed on eight positions in the MP, including two conserved sequence motifs, the LPL and D motifs. Results show that these two conserved motifs as well as the central region of the MP are essential for cell-to-cell movement. Several viruses carrying mutations in the N- or C-terminal parts of their MP retained infectivity on cowpea plants. Coexpression studies revealed that mutant MPs did not interfere with the activity of wild-type MP and could not mutually complement their defects.