In higher plant cells, vacuoles show considerable diversity in their shapes and functions. The roles of vacuoles in the storage, osmoregulation, digestion and secretory pathway are well established; however, their functions in cell morphogenesis and cell division are still unclear. To observe the dynamic changes of vacuoles in living plant cells, we attempted to visualize the vacuolar membrane (VM) by pulse-labeling tobacco BY-2 cells with a styryl fluorescent dye, FM4-64. By time-sequence observations using confocal laser scanning microscopy (CLSM), we could follow the dynamics of vacuolar structures throughout the cell cycle in living higher plant cells. We also confirmed the dynamic changes of VM structures by the observation using transgenic BY-2 cells expressing GFP-AtVam3p fusion protein (BY-GV). Furthermore, by using transgenic BY-2 cells that stably express a GFP-tubulin fusion protein [BY-GT16, Kumagai et al. (2001) Plant Cell Physiol. 42: 723], we could study the relationship between the dynamics of vacuoles and microtubules. From these observations, we identified, for the first time, some remarkable events: (1) at the late G(2) phase, tubular structures of the vacuolar membrane developed in the central region of the cell, probably in the premitotic cytoplasmic band (phragmosome), surrounding the mitotic apparatus; (2) from anaphase to telophase, these tubular structures invaded the region of the phragmoplast within which the cell plate was being formed; (3) at the early G(1) phase, some of the tubular structures expanded rapidly between the cell plate and daughter nuclei, and subsequently developed into large vacuoles at interphase.