We investigated the in situ localization of the 50 kDa protein encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) genome which is thought to be a movement protein. In immunogold electron microscopy of ACLSV-infected Chenopodium quinoa leaves, the 50 kDa protein was localized on plasmodesmata and nearby cytoplasm. Observation of transgenic Nicotiana occidentalis leaves expressing the 50 kDa protein fused to enhanced green fluorescent protein (EGFP) by fluorescence and confocal laser scanning microscopes revealed that green fluorescence was observed as spots on the cell wall or strands passing through the cell wall of several cell types, i.e., epidermal, palisade and spongy mesophyll and collenchyma cells. In transverse and longitudinal sections of leaf veins of transgenic plants showed that the 50K-EGFP fusion accumulated in sieve elements and formed an extensive interconnecting network of threadlike structure. These results indicated that ACLSV 50 kDa protein can target plasmodesmata and traffic into sieve elements.