The 25-kDa protein of potato virus X (PVX) is encoded by the 5' open reading frame of the overlapping triple gene block, has a GKS sequence motif characteristic of nucleotide binding proteins, and is thought to play a role in movement between cells. We have produced a polyclonal antibody to the 25-kDa protein to investigate its synthesis and intracellular location. On Western blots of infected tissue extracts this antibody detected a single major protein of the expected mobility, produced earlier than the viral coat protein in infected protoplasts and located predominantly in the 1000 g pellet fraction of infected cells. Immunogold labeling of thin sections of infected tissue revealed that the 25-kDa protein is associated with the cytoplasmic inclusion bodies characteristic of PVX infections, but not with the cell wall or plasmodesmata. Therefore, although mutational studies indicate a role for the 25-kDa protein in the cell to cell movement of viral infection it is unlikely to act in precisely the same manner as the other well-characterized movement proteins of tobacco mosaic and cowpea mosaic viruses.