TONSOKU(TSK)/MGOUN3/BRUSHY1 from Arabidopsis thaliana, which plays an important role in the maintenance of meristem organization, contains an LGN repeat motif similar to that found in animal proteins involved in asymmetric cell division. One protein that interacts with the LGN motif of TSK in a yeast two-hybrid screen, TSK-associating protein 1 (TSA1), contains a 10-fold repeat of a unique 41 amino acid sequence. The repeat sequence, with a glutamic acid-phenylalanine-glutamic acid (EFE) conserved core sequence, is enriched with acidic amino acids. TSA1 also contains an N-terminal putative signal peptide and it interacts with the LGN motif of TSK through a C-terminal region separated from the EFE repeats by a putative membrane-spanning region. The recombinant protein consisting of EFE repeats was rich in alpha-helical structure and possessed Ca2+-binding activity. Unlike nuclear localization of TSK, the TSA1 fused with green fluorescent protein (GFP) expressed in tobacco BY-2 cells was localized in small cytoplasmic vesicles during interphase. However, cellular localization of both TSA1-GFP and GFP-TSK changed dynamically during mitosis. In particular, both GFP-TSK and TSA1-GFP were concentrated in limited areas that are close to the ends of spindle microtubules ahead of separating chromatids. These results are discussed in terms of the possible involvement of TSK and TSA1 in mitosis.